Team:UCLA/Notebook/Spider Silk Genetics/14 July 2015
Contents
7/14/2015
ICA for 9-mer Again
- We decided to try ICA for 9-mer again, with modification of using more terminator.
- Use 1 ul of 5 uM terminator, rather than 1 uL of 50 nM terminator (100-fold increase)
- Also increased volume of washes from 50 uL to 100 uL.
- Incubation times for ligation steps were reduced to 5 minutes.
- Performed ICA using 50 ng of each DNA species, and 1 uL of 5 uM of the relevant cap.
- Eluted in 15 uL of 0.01% Tween-20.
PCR for ICA 9-mer Amplification
- PCR amplified using eluate as template
- Used 0.5 uL of undiluted and 1:100 dilution.
- NO GC enhancer
- Tested annealing temperature.
| Volume (uL) | |
|---|---|
| 5x Q5 Buffer | 5 uL |
| F-03 | 1.25 uL |
| G-03 | 1.25 uL |
| dNTPs | 0.5 uL |
| Template | 0.5 uL |
| ddH2O | 16.25 |
| Q5 Polymerase | 0.25 uL |
| Total | 25 uL |
| 98 C | 30 sec |
|---|---|
| 98 C | 10 sec |
| 63.5, 66 C | 20 sec |
| 72 C | 15 sec |
| repeat from step 2 | 20x |
| 72 C | 2 min |
| 12 C | hold |
Results
- Cast 1.5% TAE gel to visualize results. Used 100 bp ladder.
Fig. 1 PCR amplification of ICA 9-mer. This time, the results are much cleaner than the one from 7/13/2015. The anticipated product corresponding to a successful 9-mer is 1018 bp.
- It seems that we are able to get ICA to work properly.
- While the accessory bands are still present to some degree, the major product is our desired 9-mer.
- We still do not know if the accessory bands are an artifact of PCR or ICA.
- It seems that a 1:100 dilution of the crude eluate does not provide sufficient template for amplification.
Gel Purification
- The bands corresponding to the 9-mer were excised, then gel extracted using qiagen kit.
- Yielded ~ 50 ng/uL in 10 uL.