LABBOOK
July 8th
1. Prepare LB solid medium.
2. Mix 500ul X-gal(20mg/ml) and 150ul IPTG(24mg/ml) together, then coat the LB agar plates with 52ul mixture each.
3. Transfer
(1) Incubate E.coli Competent Cells JM109 in ice-bath until it melt.
(2) Add 2ul M13mp18RF DNA (Has been diluted by sterile water to 10 times volume before using)into 25ul cell suspension. Shake the centrifuge tubes gently to mix the contents.
(3) then incubate in ice-bath for 30 minutes
(4) Put the centrifuge tube in 42℃ water for 90 seconds. then quickly put it in ice-bath for 2 minutes.
(5) Add 500uL LB culture medium into the centrifuge tube, then cultivate at 37 ℃/180 rpm for 1hour to revive the cells and make related gene express.
(6) Coat 100ul suspension on the X-gal and IPTG pre-coated LB agar plate, incubate the LB agar plate at 37 °C overnight.
July 9th
1. Transformation result
Figure 7-1
It is indicated in the photograph that there are blue bacterial colonies on three plates surface,showing that the transformation was successful. However, no single colony is available for subsequent experiment
2. Streak transformed JM109 E.coli cells on the pre-warmed at 37°C LB agar plate to obtain single colonies. Incubate the LB agar plate overnight at 37 °C.
Figure 7-2
3. After overnight incubation, pick 3 blue single colonies from the LB agar plate and use to inoculate a 20 mL LB culture, then Incubate for 8~12 hours at 37 °C /250 rpm
4. Transfer 2ml bacteria suspension to new LB culture medium(50ml). then incubate for 10 hours at 37 °C/250 rpm.
5. After 10 hours of oscillating, the cultures were turbidly yellow. Store them at 4 °C.
July 11th
1. Extract M13 bacteria phage
2. Extract M13 DNA
In this stage, we add too much sample to each micro centrifuge-tube(about 7ml),which may cause the M13DNA we extract contain too much impurity
3. Measure the concentration and verification of M13 DNA
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
(2) 1% agarose electrophoresis for 110V and 20min.
Figure 7-3 Electrophoretic validation of M13 DNA
As the figure 7-3 shows, there is no expected band for M13mp18 ssDNA. We analyzed several possible causes: too much impurity in the extracted DNA, transformation result is seemingly positive but virtually negative, too much sample was to be added.
July 12th
1. prepare and sterilize LB culture-medium
2. Make LB agar plate and streak X-gal and IPTG
3. Streak transformed JM109 E.coli cells(July 9 th ) on the pre-warmed at 37°C LB agar plate to obtain single colonies. Incubate the LB agar plate overnight at 37 °C
July 13th
1. Transfer ：We transfer the M13 bacteria phage RF dsDNA into JM109 Escherichia coli, and incubate transferred E.coli on LB agar plates at 37℃ upside down for 18 hours.
2. Inoculate：pick seven blue single colonies from the LB agar plate and use to inoculate seven 50 mL LB culture. Incubate for 9.5 hours at 37 °C /280 rpm
3. Dissolve staple：the concentration of each dissolved staple is 100μmol/L, then we take 10μL from each tube and mix some of them together：label mixed hairpin strands as “staple(hairpin)”(the concentration is 16.67μmol/L);label mixed monomer B’s unique strands as “staple B”(the concentration is 7.14μmol/L);label mixed monomer A’s unique strands as “staple A”(the concentration is 7.69μmol/L);label mixed other common strands as “mixed 1~108”(the concentration is 926nmol/L).
July 14th
1. Streak transformed JM109 E.coli cells on the pre-warmed at 37°C LB agar plate to obtain single colonies. In this stage, we change pipette tips in every side, so we can get single colonies(as the LB agar plates are too thin, they are broken) Incubate the LB agar plate 23hours at 37 °C. there are single colonies can be seen.
Figure 7-4
2. Extract M13 bacteria phage
After this stage, we re-suspend bacteria phage of each tube using 3ml TE buffer
3. Extract M13 DNA
the final volume is 450μL
4. Measure the concentration and verification of M13 DNA
(1) Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
(2) 1% agarose electrophoresis for 110V and 30min.
Figure 7-5
From the A260/A230 and A260/A280, we know that our DNA has too much impurity and the concentration is too low. From the picture 7-2, we think M13 DNA has been degraded, so we discard this sample.
July 16th
1. Prepare 1ml 20mg/ml X-gal and 1ml 24mg/ml IPTG
2. Mix 500ul X-gal(20mg/ml) and 150ul IPTG(24mg/ml) together, then coat the LB agar plates with 52ul mixture each.
3. Transfer
(1) Incubate E.coli Competent Cells JM109 in ice-bath until it melt.
(2) Add 2ul M13mp18RF DNA (we extracted on July 11 th and July 14 th )into 25ul cell suspension. Shake the centrifuge tubes gently to mix the contents.
(3) then incubate in ice-bath for 30 minutes
(4) Put the centrifuge tube in 42℃ water for 90 seconds. then quickly put it in ice-bath for 2 minutes.
(5) Add 200uL LB culture medium into the centrifuge tube, then cultivate at 37 ℃/180 rpm for 1 hour to revive the cells and make related gene express.
(6) Coat 100ul suspension on the X-gal and IPTG pre-coated LB plate, incubate the LB agar plate at 37 °C for 18hours
4．Inoculate：pick five blue single colonies from LB agar plate(we streaked on July 14th) and use to inoculate five 50 mL LB cultures. Incubate for 12hours at 37 °C /280 rpm
July 17th
1. Extract M13 bacteria phage
In this stage, we use the bacteria we prepared on July 16 th to extract M13 bacteria phage. At first, we centrifuge at 6000rpm for 20 minutes, and the result is not so good, so we centrifuge at 6000g for 10minutes again, and go on. finally, we get 20ml bacteria phage to extract M13 DNA(mark as A) and 20ml bacteria phage to conserve.
2. Extract M13 bacteria phage using bacteria transferred M13 bacteria phage
(1)pick white single colony from LB agar plate and use to inoculate 6ml 2×YT culture(no MgCl 2 ). Incubate for 12hours at 37°C/280rpm
(2)after that, add 4ml bacteria to 150ml 2×YT culture(with 0.005mol/L MgCl 2 ).Incubate for 2hours at 37°C/280rpm
(3)add 15ml conserved M13 bacteria phage(on July 17 th ) to bacteria and incubate for 4hours at37°C/280rpm
(4)use this suspension to extract M13 bacteria phage
(5)finally, we get 10ml M13 bacteria phage to extract M13 DNA(mark as B)
3. Extract M13 DNA
4. Measure the concentration and verification of M13 DNA
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
A260/A280 is in the normal range, which indicate that there is little protein impurity. A260/A230 is a little high but we think it’s acceptable
(2)1% agarose electrophoresis for 110V and 30min.
Figure 7-6 Electrophoretic validation of M13 DNA
From the figure 7-6, we know that the M13 DNA we extracted on July 11 th is too thin, and on July 17 th are acceptable. We use M13 DNA A to sequence and the result is positive.
5. Design PCR program
(1)structure A Lid Control Mode: reheating at 99°C
(2)structure B Lid Control Mode: reheating at 99°C
July 18th
prepare 300ml 2×YT culture(with 0.005mol/L MgCl 2 )and 350ml LB culture
July 20 th
1.Prepare 100ml TBE buffer and store at 4°C
2.Prepare 50ml folding buffer(5mM Tris-base ,1mM EDTA) that we use to synthesize DNA Origami
July 22th
1. Extract M13 bacteria phage using bacteria transferred M13 bacteria phage
(1)pick white single colony from LB agar plate and use to inoculate 6ml 2×YT culture(no MgCl 2 ). Incubate for 12hours at 37°C/280rpm
(2)after that, add 4ml bacteria to 150ml 2×YT culture(with 0.005mol/L MgCl 2 ).Incubate for 2hours at 37°C/280rpm
(3)add 15ml conserved M13 bacteria phage(on July 17 th ) to bacteria and incubate for 4hours at37°C/280rpm
(4)use this suspension to extract M13 bacteria phage
(5)finally, we get 9ml M13 bacteria phage to extract M13 DNA
July 23th
1.Extract M13 DNA
In this stage, we use M13 bacteria phage we extracted on July 22th to extract M13 DNA and get 400μL M13 DNA suspension
July 29th
1. Measure the concentration and verification of M13 DNA
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
(2)0.8% agarose electrophoresis for 110V and 30min
Figure 7-7 Electrophoretic validation of M13 DNA
From the figure 7-7, We know that M13 DNA we extracted on July 23 th is too thin, so we discard it.
2. Calculate and design the PCR reaction system
(1)the concentration of scaffold DNA: take A(July 17 th )for example, the real concentration is 131.8
×20=2636μg/ml, that is 
", then we diluted it to 50nmol/L using
TE buffer
(2)the PCR reaction system
July 30th
1. PCR reaction of monomer B
In this stage, we use the PCR program we designed on July 17 th , that is
the reaction system is as follow：
We start this PCR program at 18:00
July 31th
1. Verify the structure of monomer B
2% agarose gel electrophoresis for 110V and 30min and the electrophoresis tank is put in ice to keep cool
Figure 7-8 Electrophoretic validation of monomer B
there is no band between 4000bp to 7000bp, so we failed to yield sufficient monomer to produce visible band.
But we think it may due to the high concentration of agarose gel, so we do as follow to verify our thought.
2% agarose gel electrophoresis for110V and 30min
Figure 7-9
the two samples are marker and M13 DNA, we find that the bands are not clear, so we confirm that it’s the problem of concentration of agarose gel.
2. PCR reaction of monomer B
(1)PCR program
(2)PCR folding buffer(the ddH2O refers to double distilled water)
①buffer A:10×TAE(1L)
②buffer B:1MTris-HCl(1L,PH=8.0,T=25℃),dilute to 100mM for use
③buffer C:10×TE(1L)
④buffer D:5×folding buffer(1L)
⑤buffer E: PCR buffer:
⑥buffer F:5×Tris-EDTA
(3)PCR reaction system(total volume is 50μv)
We start this PCR program at 18:00
August 1st
1. Verify the structure of monomer B we synthesized on July 31th
(1) 0.8% agarose gel electrophoresis for 110V and 30min
(2) 1% agarose gel electrophoresis for 110V and 30min
(3) 1.5% agarose gel electrophoresis for 110V and 30min
From the figures we know that the 1.0% agarose gel electrophoresis has the brightest band so we choose this concentration of agarose gel to do the next experiments. But there is no clear band for DNA Origami, we think the PCR program has some problems, it should not have cycles
2. PCR reaction of monomer B
(1) PCR program
(2) PCR reaction system(total volume is 20 渭L ,the concentration of MgCl2 in each PCR tube is 14mM)
August 2nd
1. Verify the structure of monomer B we synthesized on August 1st
1% agarose gel electrophoresis for 110V and 30min
(1) 0.8% agarose gel electrophoresis for 110V and 30min
there is no band of monomer. there might be something wrong with our PCR program or the M13 DNA.
August 3rd
1. Review the labbook to find out our mistake
the result of nucleic acid analyzer doesn,t need to multiple by 20 because if we have set the system of sample, the apparatus would multiple by itself, that is, the detected data is the real concentration. For example, we detect the concentration of M13 we extracted from bacteria phage was 131.8ug/ml using nucleic acid analyzer, so 
, so we need to add 9μ to each 50μt reaction system to make the final concentration of scaffold 10nM. But before we thought it was 
, which is 20times more than the real data.
2. PCR reaction of monomer B
(1) PCR program is the same as which we used on August 1st
(2) PCR reaction system(total volume is 50μL ,the concentration of MgCl2 in each PCR tube is 14mM)
3. Streak transformed JM109 E.coli cells on the pre-warmed at 37℃ LB agar plate to obtain single colonies. Incubate the LB agar plate at 37℃ for 24hours.
August 4th
1. Verify the structure of monomer B we synthesized on August 4th
1% agarose gel electrophoresis for 110V and 30min
From the figure 8-5, we can see that the last six PCR buffer are better, so our next task is to test which buffer is the best and the optical concentration of MgCl2
2. Purification and concentration of monomer
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the monomer B. In this stage, we don,t control the centrifuging temperature to 4℃ but conducted this centrifugation during room temperature! So the monomer is degraded.
3. Inoculate: pick a blue single colony from the LB agar plates and use to inoculate a 50 mL LB culture, then Incubate for 8 hours at 37℃ /280 rpm. we do this in the morning and evening respectively. In the morning, the LB agar plates we using are that we streaked on July 14th and evening on August 3rd
4. Extract M13 bacteria phage
Finally, we get 40ml M13 bacteria phage for conservation and 20ml M13bacteria phage to extract M13DNA(mark as C).
August 5th
1. Extract M13 bacteria phage
We extract M13 bacteria phage using bacteria suspension we culture on yesterday evening. In this stage, we find when centrifuging, the type of centrifuge tubes must be identical. Otherwise, some tube may be ruptured. After that, we get 20ml M13 bacteria phage to extract M13 DNA (mark as D)
2. Extract M13 DNA
the M13 DNA we extract from bacteria phage C is marked as C and from bacteria phage D is marked as D
3. Measure the concentration and verification of M13 DNA
(1) Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
(2) 1% agarose gel electrophoresis of M13 DNA
From figure 8-6 & 8-7 and the data of nucleic acid analyzer, we know that the concentration and verification of these two samples are satisfying and they both can be used as scaffold.
4. PCR reaction
(1) PCR program of monomer B
(2) PCR reaction system
August 6th
1. Prepare 700ml LB culture medium,300ml 2×YT culture medium(no MgCl2)and 100ml 2×YT culture medium(with 0.005M MgCl2),then sterilize
2. Purification and concentration of monomer B:
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the monomer B
2. Extract M13 DNA
the M13 DNA we extract from bacteria phage C is marked as C and from bacteria phage D is marked as D
3. Verify the structure of monomer B we synthesized on August 5th
1% agarose gel electrophoresis for 110V and 30min
From figure 8-8 and 8-9, with the same buffer, the systems containing 10mM MgCl2 and 12 mM MgCl2 can always produce brighter bands. So we think the optical concentration for MgCl2 is 10mM or 12mM.So we just concentrate the monomer with 10mM MgCl2 or 12 mM MgCl2.In addition, with the same concentration of magnesium, buffer D has brighter bands than other buffers. Correspondingly we choose the buffer D and 10 mM MgCl2 to do the next experiment.
August 7th
1. Extract M13 bacteria phage using bacteria transfered M13 bacteria phage
(1) pick white single colony from LB agar plate and use to inoculate 6ml 2×YT culture(no MgCl2). Incubate for 13hours at 37℃/280rpm
(2) after that, add 4ml bacteria to 150ml 2×YT culture(with 0.005mol/L MgCl2).Incubate for 2hours at 37℃/280rpm
(3) add 15ml conserved M13 bacteria phage(on August 4th ) to bacteria and incubate for 4hours at 37℃/280rpm
(4) use this suspension to extract M13 bacteria phage
(5) finally, we get 10ml M13 bacteria phage to conserve
2. Inoculate: pick six blue single colonies from LB agar plate and use to inoculate six 50 mL LB cultures. Incubate for 12hours at 37℃/280 rpm
August 8th
1. Extract M13 bacteria phage
2. Extract M13 DNA
In this stage, the concentration of M13 DNA is too low, so we discard it.
August 9th
1. Transform competent E.coli with M13 DNA.
2. Inoculate：Pick a single blue colony from the LB agar plate and inoculate to a 50 ml LB culture. Incubate for 8 hours at 37 °C /280 rpm.
August 10th
1. Extract M13 bacteria phage
2. Extract M13 DNA
3. Measure the concentration and verification of M13 DNA
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
(2)1% agarose electrophoresis for 110V and 30min
Figure 8-10 Electrophoretic validation of M13 DNA
From figure 8-10 and the data of nucleic acid analyzer, we know that the concentration and verification of this two samples are satisfying and they both can be used as scaffold.
4. PCR reaction
(1)PCR program
Program (1)
Program (2)
(2)PCR reaction system(total volume is 50μL ,the concentration of MgCl2 in each PCR tube is 10mM)
August 11th
1.Verify the structure of monomer B we synthesized on August 10th Using 1% agarose gel electrophoresis to detect the structure of monomer B. In this stage, we create four different electrophoresis. .
Figure 8-11 Electrophoretic validation of monomer B
Figure 8-12 Electrophoretic validation of monomer B
Figure 8-13 Electrophoretic validation of monomer B
Figure 8-14 Electrophoretic validation of monomer B
the electrophoresis condition of the four figures are as follow：
From the figure 8-11~8-14, we know that the better PCR program is program(1) and the optical ratio of concentration of staple and scaffold is 10:1. In addition, we also can see that the better condition of electrophoresis is 110V voltage and TAE buffer as electrophoresis buffer.
2. Inoculate：Pick six single blue colonies from the LB agar plate and inoculate to six 50 ml LB culture. Incubate for 8 hours at 37 °C / 280 rpm
August 12th
1. Purification and concentration of monomer B： We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the monomer B we synthesized using PCR program (1) on August 11th
2.Extract M13 bacteria phage
3.Extract M13 DNA
4.Measure the concentration and verification of M13 DNA
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
(2)1% agarose electrophoresis for 110V and 30min.
Figure 8-15 Electrophoretic validation of M13 DNA.
We mark concentrated monomer B as *. the ratio stands the staple/scaffold of each sample. the M13 DNA is we extracted on August 12th. From figure 8-15, we know that the M13 DNA can be used as scaffold and the optical ratio of staple/scaffold is 5:1. But we think there may be broken during concentration, combining with figure 8-11 to figure 8-14, we choose 10:1 to do the next experiments.
5.Inoculate: Pick six single blue colonies from the LB agar plate and inoculate to six 50 ml LB culture. Incubate for 8 hours at 37 °C / 280 rpm.
August 13th
1.Extract M13 bacteria phage
2.Extract M13 DNA
3.Measure the concentration and verification of M13 DNA
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow.
Figure 8-16 Electrophoretic validation of M13 DNA.
From figure 8-16 and the data of nucleic acid analyzer, we know that the concentration and verification of these two samples are satisfying and they both can be used as scaffold.
August 14th
1. PCR reaction
（1）PCR program
（2） PCR reaction system(total volume is 50μL)
【caution】A1 and A12 staples are diluted to 1μmol/L
August 15th
1.Folding dimer: mix 20μL monomer A and 20μL monomer B together and incubate at 37°C for 30min,then cool to 15°C by 3en coo minute
2.Opening hairpin(transform the dimer from flexible to rigidity)：Mix 40μL dimer and 40μL 1μmol/L fuel strands and incubate at 37°C for 30min,then cool to 15°C by 3 15°C minute.
3.Closing hairpin(transform the dimer from rigidity to flexible)：mix 20μL dimer with opening hairpin add 20 μL 1μmol/L antifuel strands, incubate at 37°C for 30min, then cool to 15°C by 3hen co minute .
4.the verification of structure：1% agarose gel electrophoresis .
Figure 8-17 Electrophoretic validation of DNA Origami.
From figure 8-17, we know in some aspect that we have synthesized our structure.
5. the purification and concentration of monomers,We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers.
6. AFM imaging:In this stage, AFM has some problems, then our experiment is stopped. We find that during imaging we should keep the coverslip clean, otherwise, the background of image would be dirty.
August 19th
Inoculate：pick six blue single colonies from LB agar plate and use to inoculate six 50 mL LB cultures. Incubate for 12hours at 37 °C /280 rpm.
August 20th
1.Extract M13 bacteria phage.
2.Extract M13 DNA.
3.Measure the concentration and verification of M13 DNA.
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow.
From the data, we know the concentration and verification of M13 DNA 1and 2 can’t reach our requirement, so we discard them.
4.Inoculate: pick six blue single colonies from LB agar plate and use to inoculate six 50 mL LB cultures. Incubate for 12hours at 37 °C /280 rpm.
August 21st
1. Extract M13 bacteria phage
2.Extract M13 DNA
3.Measure the concentration and verification of M13 DNA
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow.
From the data, we know the concentration of M13 DNA 2 can’t reach our requirement, so we discard it.
(2)1% agarose electrophoresis for 110V and 30min.
Figure 8-18 Electrophoretic validation of M13 DNA
Electrophoretic validation of M13 DNA we extracted recently.
August 23rd
Inoculate: pick six blue single colonies from LB agar plate and use to inoculate six 50 mL LB cultures. Incubate for 12hours at 37 °C /280 rpm.
August 24th
1.Extract M13 bacteria phage.
2.Extract M13 DNA.
3.Measure the concentration and verification of M13 DNA.
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow.
(2)1% agarose electrophoresis for 110V and 30min.
Figure 8-19 Electrophoretic validation of M13 DNA.
Electrophoretic validation of M13 DNA we extracted recently. the M13 DNA we extracted on August 24th is too thin, so we discard it.
August 25th
1. PCR reaction
(1)PCR program:PCR program is the same as which we used on August 14th and we folding monomer A and monomer B in the same time.
(2)PCR reaction system(total volume is 50μL)
2.the purification and concentration of monomers:We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers.
(1)Insert the Amicon Ultra-0.5 device into one of the provided micro-centrifuge tubes.
(2)Add up to 100 μL of sample and 400μL PCR buffer with Mg2+ to the Amicon Ultra filter device.
(3)Spin the device at 4500 × g for 15 minutes and discard the liquid in the micro-centrifuge tube.
(4)Place the filter device upside down in the micro-centrifuge tubes, spin for 4 minutes at 1000 x g to transfer the concentrated sample from the ultra filter device to the tube.
(5)Repeat steps 2 and 3 for 3 times.
(6)Place the filter device upside down in the micro-centrifuge tubes, spin for 4 minutes at 1000 x g to transfer the concentrated sample from the ultra filter device to the tube. In this stage, we made a small mistake. After the first centrifugation, we collected DNA Origami. this mistake made no influence on our next experiment
3.Folding dimer: mix 25μL monomer A and 25μL monomer B(been centrifuged) together and incubate at 37°C for 30min,then cool to 15°C by 3en coo minute
4.Opening hairpin(transform the dimer from flexible to rigidity)：Mix 40μL dimer and 40μL 1μmol/L fuel strands and incubate at 37°C for 30min,then cool to 15°C by 3en coo minute
5.Closing hairpin(transform the dimer from rigidity to flexible)：mix 20μL dimer with opening hairpin add 20 μL 1μmol/L antifuel strands, incubate at 37°C for 30min, then cool to 15°C by 3hen co minute
6.the verification of structure：1% agarose gel electrophoresis
Figure 8-20 Electrophoretic validation of DNA Origami
We mark concentrated DNA Origami as*. From figure 8-20, we know that the DNA Origami we synthesized on August 25th are accurate in some aspects. So we use these samples to do TEM imaging.
7.Prepare 0.1μg/ml poly-L-lysine
8.Prepare samples for TEM imaging
9.TEM imagin:In this stage, we use 2% phosphotungstic acetate to stain and the working voltage is 200kV.
Figure 8-21 TEM image of dimer
From the images, we can confirm that the structure we folded is correct. But the background of our image is not clear and the profile of structure is not apparent. We think the background is due to the staining agent and the structure’s profile is due to high voltage.
August 27th
1.Extract M13 bacteria phage
2. Extract M13 DNA
3. Measure the concentration and purification of M13 DNA
(1)Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
(2)1% agarose electrophoresis for 110V and 30min
4. PCR reaction
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A and monomer B in the same time
(2)PCR reaction system (total volume is 50μL)
5. the purification and concentration of monomers
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
6. Folding dimer: mix 20μL monomer A and 20μL monomer B together and incubate at 37°C for 30min,then cool to 15°C by 3en coo minute
7. Opening hairpin(transform the dimer from flexible to rigidity)：Mix 40μL dimer and 40μL 1μmol/L fuel strands and incubate at 37°C for 30min,then cool to 15°C by 3℃ per minute
8. Closing hairpin(transform the dimer from rigidity to flexible)：mix 20μL dimer with opening hairpin add 20 μL 1μmol/L antifuel strands, incubate at 37°C for 30min, then cool to 15°C by 3℃ per minute
9. Measurement of fluorescent intensity changes
We measure the fluorescent intensity changes by Quanta Master™ 400 fluorescence spectrophotometer using a quartz cuvette at room temperature about 20°C.
Figure 8-22 fluorescent intensity measurement
there is little fluorescent intensity at 650~700nm, the signal of FRET is not apparent. through reading some literatures, we know that the valid reaction distance of cy3 and cy5 is less than 7.2nm, but in our structure, their distance is 9.25nm(closing hairpin) and 14.25nm (opening hairpin)respectively. So we redesign the staple A12 to reduce the distance of cy3 and cy5 to 3.7nm when closing hairpin theoretically
August 28th
1. PCR reaction
PCR program and reaction system are as the same as which we used on August 27th and we start the PCR reaction at 17：45
2. the purification and concentration of monomers
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
3. Folding dimer
4. Opening hairpin(transform the dimer from flexible to rigidity)
5. Closing hairpin(transform the dimer from rigidity to flexible)
August 29th
1. the verification of structure we synthesized on August 28th.
1% agarose gel electrophoresis for 110V and 30min
Figure 8-23 Electrophoretic validation of DNA Origami
We mark concentrated DNA Origami as *. From figure 8-23&8-24, we know that concentration can increase the concentration of DNA Origami. Consequently, we use concentrated DNA Origami to do subsequent experiments.
2. AFM imaging
(1)Because time is tight, we dip the coverslip in 0.1mg/mL poly-L-lysine only for 2hours
(2)the AFM has some problems causing that we can’t continue our AFM imaging
August 31th
1. PCR reaction
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A and monomer B in the same time
(2)PCR reaction system(total volume is 50μL)
September 1st
1. the purification and concentration of monomers
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
2. Folding dimer
3. Opening hairpin(transform the dimer from flexible to rigid=ity)
4. Closing hairpin(transform the dimer from rigid=ity to flexible)
5. Rheology measurement
In this stage, we use KINEXUS rotational rheometer to detect the viscosity changing.
(1)Ensure the proper shear rate: we use monomer B to operate this experiment
Figure 9-1: Shear viscosity-shear rate
From fig 9-1, we know when shear rate is more than 10s-1 ,the viscosity couldn’t change any more ,but in today’s next experiment, we use 100s-1.
(2)Measure the viscosity：we use 100s-1shear rate to detect shear viscosity transformation
Figure 9-2 viscosity measurement: (A)viscosity comparation of dimer added fuel strands and dimer added fuel strands and antifuel strands (B)shear viscosity of dimer added fuel strands subtracts which of dimer added fuel strands and antifuel strands(C)histogram of sample data of different shear viscosity(viscosity of dimer added fuel strands subtracts which of dimer added fuel strands and antifuel strands) D-value
From fig 9-2, the shear viscosity has little changes between the two dimers and the values are too low to compare, we think it may because the concentration and the degree of polymerization of the samples are too low and the shear rate is too high
September 5th
1. Inoculate：pick six blue single colonies from LB agar plate and use to inoculate six 50 mL LB cultures. Incubate for 12hours at 37 °C /280 rpm
September 6th
1. Extract M13 bacteria phage
2. Extract M13 DNA
3. Measure the concentration and verification of M13 DNA
Use the nucleic acid= analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
4. PCR reaction
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A and monomer B in the same time
(2)PCR reaction system(total volume is 50μL)
5. the purification and concentration of monomers
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
6. Folding dimer
7. Opening hairpin(transform the dimer from flexible to rigid=ity)
8. Closing hairpin(transform the dimer from rigid=ity to flexible)
9. Prepare samples for TEM imaging
September 7th
TEM imaging of dimer
In this stage, we use 2% uranyl acetate to stain and the working voltage is 70kV.
Figure 9-3 TEM image of monomer
Figure 9-4 TEM image of dimer
From the images, we can confirm that the objects we folded are correct. the object is complete and background of our image is clear. Because of the high concentration of our DNA Origami, some monomers and dimers have overlaps with each other.
September 11th
1. PCR reaction
PCR program
PCR program is the same as which we used on August 14th and we folding monomer A and monomer B in the same time
(2)PCR reaction system(total volume is 50μL)
2. the purification and concentration of monomers
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
3. Folding dimer
4. Opening hairpin(transform the dimer from flexible to rigid=ity)
5. Closing hairpin(transform the dimer from rigid=ity to flexible)
September 12th
1.Measurement of fluorescent intensity changes.
We measure the fluorescent intensity changes by Quanta Master™ 400 fluorescence spectrophotometer using a quartz cuvette at room temperature about 20°C. Donor (Cy3) fluorescence is excited by illumination at 532 nm, and detect the fluorescent intensity changes with wavelength
Figure 9-5 Fluorescent changes of DNA Origami
there is no apparent FRET phenomenon in our experiment, but when we use 646nm wave to excite, the emission of cy5 can be detected. So two staples have been combined to DNA Origami, no fret may because we make a mistake of ensuring the newly synthesized A12 staple.
2. Rheology measurement
We use 10s-1shear rate to detect shear viscosity transformation. In this stage, we detect the viscosity changes of different degree of polymerization and different rigidity statements
Figure 9-6 Rheology measurement: (A)viscosity comparation of dimer added fuel strands and dimer added fuel strands and antifuel strands (B)histogram of sample data of different shear viscosity(viscosity of dimer added fuel strands subtracts which of dimer added fuel strands and antifuel strands) D-value(C)shear viscosity of dimer subtracts which of dimer added fuel strands and antifuel strands (D)histogram of sample data of different shear viscosity(viscosity of dimer subtracts which of dimer added fuel strands and antifuel strands) D-value
From fig 9-6&fig 9-7,we know that both degree of polymerization and object rigidity can result in changing of viscosity, if we could increase the concentration and degree of polymerization, it may have obvious transformation in viscosity, which can use in alter blood viscosity.
September 17th
1. Prepare electroporation buffer.
Dissolve 0.585g NaCl,0.1615g MgCl2, 18.217g mannitol in 1L 10Mm HEPES.
2.PCR reaction
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A and monomer B in the same time
(2)PCR reaction system(total volume is 50μL)
3.the purification and concentration of monomers
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
4.Folding dimer
5.Opening hairpin(transform the dimer from flexible to rigidity)
6.Closing hairpin(transform the dimer from rigidity to flexible)
September 18th
1. H22 cell preparation.
(1)Centrifuge H22 cell at 1000rpm for 3min and discard the supernatant. (2)Resuspend the cell sediment by PBS buffer (3)Put 100ell sediment nsion into 1.5ml centrifuge tube and incubate on ice for 20~30 minutes (4)Centrifuge at 4000 rpm for 10 minutes at 4℃. Discard the supernatant (5)Add 100ata10% glycerol into centrifuge tube and Centrifuge at 4000 rpm for 10 minutes at 4℃. Discard the supernatant (6)Repeat step3 for three time (7)Add 100hree timestrifuge tube and leyilt then store at 4℃
2. Put DNA Origami into H22 cells and liposomes by electroporation
After electroporation, we put samples at 4°C. the parameter of electroporation: U=150v;C=25uF;R=+++;Gap=2mm;
3.Test the rigidity of H22 cells
Because the MTC has some problems, we can’t operate this experiment to test rigidity of H22 cells
September 19th
1.Prepare samples for testing rigidity of liposomes by AFM
In this stage, we use air model to test. Add 10μl sample on the sample-membrane and incubate for 20mi, then dry in the air.
September 20th
1.Extract M13 bacteria phagev
2.Extract M13 DNA
3.Measure the concentration and verification of M13 DNA
Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
4.PCR reaction
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A and monomer B in the same time
(2)PCR reaction system(total volume is 50μL)
5.the purification and concentration of monomers
6.Folding dimer
7.Opening hairpin(transform the dimer from flexible to rigidity)
8.Closing hairpin(transform the dimer from rigidity to flexible)
September 21st
1.Fluorescence microscope observation.
In this stage, we observe the objects in a magnification of 20 fold, the donor dye Cy3 on Nanoman is excited using the 559nm laser. the resulting fluorescence, Cy3’s and acceptor dye Cy5’s emitted light, is recorded in two independent channels set up to detect emitted spectra correspondingly in the ranges 570-625 nm(for Cy3) and 655-755 nm(for Cy5). Because of the long exposure time, we couldn’t observe fluorescence.
September 22nd
1.Put DNA Origami into liposomes by electroporation
In this stage, we use Gene Pulser MXcell Electroporator to operate electroporation.
2.Detect the rigidity of the liposome by AFM:
(1)sample preparation: In this stage, we use air model to test. Add 10μl sample on the sample-membrane and incubate for 20mi, then dry in the air (2)using non-contact mode to measure the physical properties of our DNA Origami, the sacn speed is 1.000Hz and operating is 0.500V, scan start voltage is 200.000V, scan range voltage is 395.605V, the scan range position is 3033.325nm~-2966.675nm. (3)after imaging, we get AFM image and the data about deflection approach and deflection release. (4)according to other information, we gain the physical properties.
3.Inoculate: pick six blue single colonies from LB agar plate and use to inoculate six 50 mL LB cultures. Incubate for 12hours at 37 °C /280 rpm
4.Rheology measurement
We use 10s-1shear rate to detect shear viscosity transformation. In this stage, we detect the viscosity changes of different rigidity statements
Figure 9-8 Rheology measurement: (A)viscosity comparation of dimer added fuel strands and dimer(B)histogram of sample data of different shear viscosity(viscosity of dimer added fuel strands subtracts which of dimer) D-value
From fig 9-8, we can see there are sometimes the D-value of viscosity are in our expectation.
September 23rd
1. Extract M13 bacteria phage
2. Extract M13 DNA
3. Measure the concentration and verification of M13 DNA
Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. the samples are diluted by 20times. the results are as follow
4. PCR reaction of monomer A
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A and monomer B in the same time
(2)PCR reaction system(total volume is 50μL)
September 24th
1. the purification and concentration of monomer A
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
2. Measurement of fluorescent intensity changes
In this stage, we want to ensure which A12 staples is valid. We measure the fluorescent intensity changes by Quanta Master™ 400 fluorescence spectrophotometer using a quartz cuvette at room temperature about 20°C. Donor (Cy3) fluorescence is excited by illumination at 532 nm, and donor emission is measured at 567nm, and acceptor emission is measured at 667nm
Figure 9-9 Fluorescent changes of DNA Origami: fluorescent intensity of monomer A synthesized by different A12 staples. A1-1 stands the first A12 staples and A1-2 stands another. At 200s, we add fuel strands to the system and regulate the concentration of these samples identical.
From fig 9-9, after adding fuel strands, the fluorescent intensity of cy5 reduces a lot, especially A11, for the disappearance of fret phenomenon and we think that the A12 staple we used in A1-1 is newly synthesized.
September 27th
1. PCR reaction
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A and monomer B in the same time
(2)PCR reaction system(total volume is 50μL)
2. the purification and concentration of monomers
3. Folding dimer
4. Opening hairpin(transform the dimer from flexible to rigidity)
5. Closing hairpin(transform the dimer from rigidity to flexible)
September 28th
1. Measurement of fluorescent intensity changes
We measure the fluorescent intensity changes by Quanta Master™ 400 fluorescence spectrophotometer using a quartz cuvette at room temperature about 20°C. Donor (Cy3) fluorescence is excited by illumination at 532 nm, and donor emission is measured at 567nm, and acceptor emission is measured at 667nm
Figure 9-10 Fluorescent changes of DNA Origami: fluorescent intensity of monomer A and dimer. At 200s, we add fuel strands to the system and regulate the concentration of these samples identical. At 400s, we add antifuel strands to the system and regulate the concentration of these samples identical
From fig 9-10, after adding fuel strands, the fluorescent intensity of cy5 reduces a little, for the disappearance of FRET phenomenon. But when add antifuel strands, there is no apparent change in fluorescent intensity, in which the cy3 should reduce and cy5 should increase in theory. So we don’t get the result in expectation.
2. Fluorescence microscope observation
In this stage, we observe the objects in a magnification of 20 fold, the donor dye Cy3 on Nanoman is excited using the 559nm laser. the resulting fluorescence, Cy3’s and acceptor dye Cy5’s emitted light, is recorded in two independent channels set up to detect emitted spectra correspondingly in the ranges 570-625 nm (for Cy3) and 655-755 nm (for Cy5).
Figure 9-11 Fluorescence imaging of the real-time FRET ： a) Two different conformation of Nanomen monomer, corresponding to FRET status (top) and non-FRET status (bottom) respectively. b) Images taken by LSCM. the top three images are taken from the same sample with FRET effect while the three images in the bottom are taken from the sample without FRET effect. the yellow spots (non-FRET Nanomen) are marked and numbered in the image.
From fig 9-11, we know that the monomer can change its conformation when adding different strands, so the external tube can move as we expected.
October 1st
1.Prepare LB culture medium.
2.Inoculate: Pick six single blue colonies from the LB agar plate and inoculate to six 50 ml LB culture. Incubate for 12 hours at 37 °C / 280 rpm.
October 2nd
1. Extract M13 bacteria phage
2. Extract M13 DNA
3. Measurement of concentration and verification of M13 DNA
Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. The samples are diluted by 20times. The results are as follow:
October 3rd
PCR reaction of monomer A
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A
(2)PCR reactionsystem(total volume is 50μL)
October 4th
1. The purification and concentration of monomer A
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
2. Measurement of fluorescent intensity changes
We measure the fluorescent intensity changes by Quanta Master™ 400 fluorescence spectrophotometer using a quartz cuvette at room temperature about 20°C and two modes to test the fluorescent intensity：time-fluorescent intensity and wavelength-fluorescent intensity. In time-fluorescent mode, donor (Cy3) fluorescence is excited by illumination at 532 nm, donor (cy3) emission is measured at 567nm, and acceptor (cy5) emission is measured at 667nm. In wavelength-fluorescent intensity mode, the excitation wavelength is 532nm.
Figure 9-14 fluorescence change of DNA origami testing in wavelength-fluorescent intensity mode.
In theory, monomer A and monomer A with fuel strands and antifuel strands can cause FRET effect, while monomer A with fuel strands can’t cause FRET effect. During the detection process, the addition of antifuel strands and fuel strands may result in loud noise, which may be a reason of the non-signal result of FRET.
Figure 9-15 fluorescence change of DNA origami testing in time-fluorescent intensity mode
During 0~200s, we detect the fluorescence intensity of monomer A, and 200~400s, monomer A with fuel strands, 400~600s, monomer A with fuel strands and antifuel strands. In the later stage of each sample, the intensity of Cy3 has a little enhancement, which demonstrate the loud noise caused by other strands.
October 5th
1. Prepare LB culture medium.
2. Inoculate: Pick six single blue colonies from the LB agar plate and inoculate to six 50 ml LB culture. Incubate for 12 hours at 37 °C / 280 rpm
October 6th
1. Extract M13 bacteria phage
2. Extract M13 DNA
3. Measurement of concentration and verification of M13 DNA
Use the nucleic acid analyzer to measure the concentration of M13 DNA. Use TE buffer to set zero. The samples are diluted by 20times. The results are as follow:
October 7th
PCR reaction of monomer A
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A
(2)PCR reactionsystem(total volume is 50μL)
October 8th
1. The purification and concentration of monomer A
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
2. Folding dimer
3. Opening hairpin(transform the dimer from flexible to rigidity)
4. Closing hairpin(transform the dimer from rigidity to flexible)
October 9th
PCR reaction of monomer A and monomer B
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A
(2)PCR reactionsystem(total volume is 50μL)
PCR reaction system is the same as which we used on October 7th .
October 10th
1.The purification and concentration of monomer A
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
2.Measurement of fluorescent intensity changes
We measure the fluorescent intensity changes by Quanta Master™ 400 fluorescence spectrophotometer using a quartz cuvette at room temperature about 20°C and two modes to test the fluorescent intensity: wavelength-fluorescent intensity and time-fluorescent intensity. In time-fluorescent mode, donor (Cy3) fluorescence is excited by illumination at 532 nm, donor (cy3) emission is measured at 567nm, and acceptor (cy5) emission is measured at 667nm. In wavelength-fluorescent intensity mode, the excitation wavelength is 532nm.
Figure 9-16 fluorescence change of DNA origami testing in wavelength-fluorescent intensity mode.
Besides the emission at 550nm~600nm, we also detect emission at 640nm~700 caused by the acceptor (Cy5) emission. It indicates successful structure transformation of monomer A after adding fuel strands and antifuel strands.
Figure 9-17 fluorescence change of DNA origami testing in time-fluorescent intensity mode
In figure 9-17, during 0~200s, we detect the fluorescence intensity of monomer A, and 200~400s, monomer A with fuel strands, 400~600s, monomer A with fuel strands and antifuel strands. After add fuel strands at 200s, there is an apparent enhancement of fluorescence intensity of Cy3, accompanied by a weakness of cy5 fluorescence intensity. When we add antifule strands, cy3 and cy5 recover their intensities during 0~200s.
October 12th
PCR reaction of monomer A and monomer B
(1)PCR program
PCR program is the same as which we used on August 14th and we folding monomer A
(2)PCR reactionsystem(total volume is 50μL)
PCR reaction system is the same as which we used on October 7th
October 13th
1. The purification and concentration of monomer A
We use Amicon Ultra-0.5 Centrifugal Filters to purify and concentrate the two monomers
2. Folding dimer: mix 20μL monomer A and 20μL monomer B together and incubate at 37°C for 30min,then cool to 15°C by 3en coo minute
3. Opening hairpin(transform the dimer from flexible to rigidity)：Mix 40μL dimer and 40μL 1μmol/L fuel strands and incubate at 37°C for 30min,then cool to 15°C by 3 15°C minute
4. Closing hairpin(transform the dimer from rigidity to flexible)：mix 20μL dimer with opening hairpin add 20 μL 1μmol/L antifuel strands, incubate at 37°C for 30min, then cool to 15°C by 3hen co minute
5. AFM imaging
Figure 10-5 AFM and model images of flexible dimer. (A, B) Wide field image of successfully assembled flexible dimer with a high yield. (C) A magnified image of flexible dimer (B, D) Height profile, curve between the vertical lines indicates the height of flexible dimer framed in image (E) A model image corresponding to the real structure illustrated in image C to give a vivid cognition of the successfully assembled flexible dimer. (F) The merged image of the AFM image and the designed model.
Figure 10-6 AFM and model images of rigid dimer by adding fuel strands. (A, B) Wide field image of successfully assembled rigid dimer with a high yield. (C) A magnified image of rigid dimer (B, D) Height profile, curve between the vertical lines indicates the height of rigid dimer framed in image (E) A model image corresponding to the real structure illustrated in image C to give a vivid cognition of the successfully assembled rigid dimer. (F) The merged image of the AFM image and the designed model.
The intact structure of flexible dimer and rigid dimer is successfully folded after the origami experiment. Followed with a purification and concentration, we can finally see flexible dimer and rigid dimer under AFM with a high yield. The magnified image of flexible dimer and rigid dimer shown in figure 10-5C and figure 10-6C are corresponded to the model images in figure 10-5E and figure10-6E respectively, so that we can have a clearer understanding of their structure. In figure 10-5D and figure 10-6D, the height profile is given suggesting that flexible dimer measures about 7nm and rigid dimer measures about 28nmin height, approaching our design 6nm and 20nm respectively, which confirms the successful folding and the dimer’s conformation switch.
