Team:UCLA/Notebook/Honeybee Silk/5 May 2015
Contents
Colony PCR of 5/4 Transformation
5/4 Transformation Results
- Individual colonies were present on 1:1 plates, none/indiscernible present on 1:10 plates.
- We picked 3 colonies from each the chloramphenicol plates (silk and silk+promoter) and suspended each in an epi tube with 99 uL ddH20.
Colony PCR Reaction
- using Q5, transformed E. coli, and the VF2 and VR primers in preparation for insertion into psb1c3
- Three 25 uL reactions each for:
- Silk with VF2 and VR
- SIlk+promoter with VF2 and VR
| Component | Volume (out of 25uL) |
|---|---|
| 5X Q5 Reaction Buffer* | 5uL |
| 10mM dNTPS* | 0.5uL |
| 10mM VF2 primer* | 1.25uL |
| 10mM VR primer* | 1.25uL |
| Transformed cells in ddH2O | 1uL |
| Q5 High Fidelity DNA Polymerase | 0.25uL |
| Nuclease Free Water* | 15.75uL |
- I made a Mastermix of the starred components, then added 23.75uL of the mix to each PCR strip, followed by the corresponding transformed cells and Q5 DNA Polymerase.
| Step | Temperature | Time |
|---|---|---|
| Initial Denaturation | 98C | 3 min |
| Cycles (x25) | 98C | 10s |
| Annealing | 66C | 15s |
| Extension | 72C | 15s |
| Final Extension | 72C | 2min |
| Hold | 12C | Hold |
- TM calculated using NEB TM Calculator
- Total Run Time 35 minutes (including ramp times)
Gel Visualization of PCR Products
- 5uL of 6X loading dye was added to each 25uL PCR product
- 10uL of 1kb ladder was prepared with 2uL of 6X loading dye
I ran two 5uL samples of each PCR product against the ladder, one well was left empty between the different samples.
From left to right: Ladder, S1, S2, S3, SP1, SP2, SP3 Expected Band sizes: ~1200bp, ~1500bp File:Gel 5/5.tif
Inoculation
99uL of each sample was inoculated in 6 difference culture tubes containing 10mL LM and 1uL of 1000x chloramphenicol and incubated overnight at 37C.