Team:UCLA/Notebook/Spider Silk Genetics/19 June 2015
6/19/2015: Testing ICA
Plan for ICA
Plan on creating a 3-mer, complete with initiator, terminator. Some slight modifications from Briggs et al.
- 5 uL of Magnabind streptavidin beads.
- wash 2x using 2x BW buffer (15 uL washes).
- resuspend in 5 uL 2x BW buffer.
- Add 1 uL of 5 uM initiator and 4 uL ddH2O.
- Rotate at RT 30 min.
- Wash 2x using 0.5x BW buffer.
- set up a 10 uL ligation reaction containing the following:
- 5 uL 2x T7 Ligase Buffer
- 50 ng relevant DNA (see table below)
- 1 uL of relevant cap (5 uM concentration)
- ddH2O to 10 uL.
- Incubate at RT for 10 min.
- Repeat steps 5,6 for BC, CA and Terminator.
- Wash 1x using ddH2O.
- Elute in 15 uL of 0.01% Tween-20 at 95 C for 3 min.
- Assess ICA using e-gel and PCR amplification at different cycles (12, 15, 17, 20)
| Concentration (ng/uL) | Volume for 50 ng (uL) | |
|---|---|---|
| AB | 41.5 | 1.2 |
| BC | 51 | 0.98 |
| CA | 36.8 | 1.36 |
ICA Assessment
E-gel
- ran with David on 1% e-gel.
- used 2.5 uL of eluted sample
- used 1 uL of 100 bp ladder
- diluted all samples to 20 uL using ddH2O
- Nothing showed up. (no picture)
- Decided to go ahead with PCR anyway, since there MIGHT be something.
PCR
- Set up 4x 25 uL reactions each with 1 uL of eluted sample as the template and post-elution primers.
- Followed Q5 protocolaccording to NEB, with the addition of GC enhancer.
- We used the following cycle scheme:
| 98 C | 30 sec |
| 98 C | 10 sec |
| 66 C | 15 sec |
| 72 C | 20 sec |
| repeat from step 2 | 12, 15, 17, 20x |
| 72 C | 2 min |
| 12 C | hold |
- cast 1.5% TAE gel to visualize. Used 2 ul of 100 bp ladder.
- From the PCR, we can conclude that there was no finished ICA product.
- This problem may lie in improper bead binding, improper ligations, or incorrect PCR.
- MANY reasons why this might fail.
- Tomorrow, test the streptavidin/biotin bead binding.