Team:Cambridge/Experiments/Assembly of Reflectin Constructs
From 2011.igem.org
(Difference between revisions)
(→PCR) |
(→PCR) |
||
Line 14: | Line 14: | ||
{| border="1px" align="center" style="text-align:center;" | {| border="1px" align="center" style="text-align:center;" | ||
|Hold | |Hold | ||
+ | | | ||
|95 | |95 | ||
|2min | |2min |
Revision as of 11:37, 19 August 2011
Loading...
This is a placeholder. We should fill it in.
Contents |
Construct Design
Primer Design
We should mention expected lengths of products here.
Assembly: first attempt
PCR
In the first round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3 and GA4 constructs.
- We performed PCR using Phusion Hot Start DNA Polymerase in 20 μl reaction volume.
- The time profile used in the PCR machine was the following:
Hold | 95 | 2min | |
Cycling | Denaturing | 95 | 10s |
Annealing | 55 | 20s | |
Elongation | 72 | 150s |
- We decided to use 55 degrees annealing temperaure, although the calculated temperature for most primers is 5-10 degrees higher, because of low annealing temperature of the VF2 primer.
Gibson Assembly
Transformation
Results
Diagnostics
Assembly: second attempt
PCR
Gibson Assembly
Transformation
Results
What next?