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Team:SUSTC-Shenzhen/Notebook/CRISPR/UAS-Fill-in-The-third-time
From 2014.igem.org
Notebook
Elements of the endeavor.
UAS Fill-in (The third time)
2014/8/12~2014/8/13Materials
The same as the first Fill-in experiment.
Methods
Restriction digestion for 5*UAS plasmid with EcoRI
Digestion system：（unit: ul）
| Total volume | 10X Buffer | DNA | EcoRI | ddH2O | |
|---|---|---|---|---|---|
| Poly A(168.8ng/ul) | 30 | 3 | 15 | 3 | 9 |
| G-PB5(293.6ng/ul) | 30 | 3 | 15 | 3 | 9 |
Time: 8.11 8:20pm~ 10:00pm 8.12 9:30am~4:30pm [incubate 37°, overnight]
Electrophoresis to verify if most of the digested plasmid has been linearized
Loading system：（unit: ul）
| Total volume/well | DNA | Dye | TAE |
| 12 | 1 | 2 | 9 |
Running conditions: 120V, 35min
Results of electrophoresis:
From the picture, we can see that, the position of the band of digested plasmid is apparently lying behind that of the original plasmid which hasn’t been digested by EcoRI, indicating that most of the plasmid in our digestion system has become linearized.
Transformation for these digested plasmids, to estimate the percentage of false positive colonies.
Performed as protocol for transformation. dd 2ul digested plasmid into 50ul competent cell Incubate., 37C, overnight [8.12 9:00pm~8.13 9:30am]
Results:
No colonies have grown on any of these plates, which declare nearly all of the plasmids in our digestion system have been linearized.
Heat-inactivation for the digestion system & Cold shock to prevent self-ligation
75°C，10min and chill on ice
Do fill-in with T4 DNA polymerase
The remaining quantity of DNA in each tube
Till now, there are still 27ul reaction liquid remaining in each tube. The quantity of DNA in each tube: Poly A: 2.1788ug G-PB5: 3.9595ug
Fill-in：（unit: ul）
| Total volume | DNA | T4 DNA polymerase(3U/ul) | dNTP (10mM) | 10X NEBuffer | |
| Poly A(21.1ng/ul) | 31.1 | 27 | 0.8 (theoretical volume:0.726ul) | 0.3 | 3 |
| G-PB5(29.36ng/ul) | 31.7 | 27 | 1.4 (theoretical volume:1.132ul) | 0.3 | 3 |
[According to the manual provided by NEB：The NEBuffer 2.1 for EcoRI is also suitable for T4 DNA polymerase and, 1ug DNA ~ 1 unit enzyme; final concentration for dNTP: 100uM]
Protocol
a. incubate at 12°C，15min.
b. add 3ul EDTA(100mM) to 27ul reaction system to stop the polyreaction.
c. put in 75°C，20min to inactivate T4 DNA polymerase thoroughly.
Purification of DNA by TIANquick Purification Kit to remove EcoRI，NEBuffer，T4 DNA polymerase etc.（borrowed from our RA）
Protocol:
Performed as the protocol provided, except that we eluted the DNA sample with 68°C ddH2O twice at the last step.
Results：Using 0.5ul DNA for Nanodrop Measurement
| Concentration | 260/280 | 260/230 | |
| Poly A | 87.4ng/ul | 1.84 | 2.02 |
| G-PB5 | 41.2ng/ul | 1.87 | 1.70 |
Blunt end ligation with T4 DNA ligase [Design a concentration gradient]
Till now, there are still 29.5ul DNA remaining in each tube
Ligation system：（unit: ul）
| Poly-A | Total volume | DNA | 10X T4 DNA ligase buffer | T4 ligase | EcoRI (to reduce those sticky ends ligation) | ddH2O |
| 100ng rank | 31 | 1.5 | 3 | 1 | 25.5 | |
| 800ng rank | 31 | 10 | 3 | 5 | 13 | |
| 2000ng rank | 31 | 18 | 3 | 10 | 0 | |
| G-PB5 | Total volume | DNA | 10X T4 DNA ligase buffer | T4 ligase | EcoRI (to reduce those sticky ends ligation) | ddH2O |
| 50ng rank | 31 | 1.5 | 3 | 0.5 | 26 | |
| 400ng rank | 31 | 10 | 3 | 2.5 | 15.5 | |
| 1000ng rank | 31 | 18 | 3 | 5 | 5 |
Protocol：
a. incubate at 16°C，15h[8.12 1:30pm ~ 8.13 8:00am
b. Heat inactivation 65°C, 10min
c. chill on ice
Restriction digestion with EcoRI, to re-linearize those sticky-end-ligations
Divide the 31ul ligation reaction into two tubes, around 15ul for each
Take one of the two tubes for restriction digestion.
Reaction system [unit: ul]
| Total volume | DNA | EcoRI | 10*NEBuffer | ddH2O |
| 20 | 15 | 0.5 | 2 | 2.5 |
Protocol:
incubate 37°C，5h [2014.8.13 2:46pm ~ 19:30pm]
Heat inactivation
70°C, 10min
Transformation
Performed as protocol
1.Add 5ul DNA into 50ul competent cell. [Save the remaining 15ul DNA as backups].
2.We also transformed bacteria with those DNA that haven’t been digested by EcoRI as well. To make sure, if there were no colonies growing out, the reason shouldn’t lie in the ligation step.
3. Recover step: 200rpm, 45min.
4. Centrifuge before distributing on agar plate: 4000rpm, 5min.
5. Incubate, 37°C, for 3 days. [2014.8.13 10:22pm~ 8.14/15/16]
Results
During the following three days, no colonies have grown on any of these 12 plates…
Troubleshooting
This result is extremely strange, since even for the control group(undigested with EcoRI) there were no colonies grown out. We then do electrophoresis for those backups, in order to find the crux. To our surprise, the electrophoresis graph showed that there were no DNA plasmids or even linearized DNA fragments in the ligation reaction liquid, due to the lack of bright bands of DNA along each track.
After consulting with our RA, we speculated that: 1) the ddH2O we used had been contaminated with some bacteria which released DNAzyme, degrading our DNA in the reaction liquids. 2) There are some problems with the DNA Purification kit we used in step5. We brought it from a researcher of Prof. Yu, and he couldn’t warrant that the kit was efficacious. Even though we had detected the concentration of DNA after purification, and the values of which were reasonable (see the table in step5), we still couldn’t assert that the DNA we got was normal. Because maybe it was the dNTP remaining that contributed to the concentration we detected.
All in all, the third time Fill-in end in failure.
Contemplations
1) We should reserve a quantity of backups for each operative steps, so that if the final results show something unexpected, we can go back to check out the cruxes step by step with these reserved samples.
2) DO NOT use the DNA Purification kit the next time.
3) Make sure that the ddH2O we used each time was free off bacteria.
