"
Team:Paris Saclay/Notebook/August/7
From 2014.igem.org
Contents |
Thursday 7th August
Lab work
C - Salicylate Inducible Suppressing System
DNA extraction from gel agarose
In progress
D - Lemon Sent
PCR of pCola plasmid (Geranyl Synthase)
'by Melanie'
we use the same protocol as yesterday but we have done 8 tubes to apply a gradient in the thermocycleur during the third step:
- 57°
- 56.3°
- 55.1°
- 53.3°
- 51°
- 49.3°
- 47.9°
- 47°
PCR protocol: 98° --> 2min
5 PCR cycle:
| time | 10sec | 20sec | 45sec |
|---|---|---|---|
| temperature | 98° | (depending on the tube - see the gradient) | 72° |
25 PCR cycle
| time | 10sec | 20sec | 45sec |
|---|---|---|---|
| temperature | 98° | 72° | 72° |
and last step: 72° during 10min
but we don't have any results
Test the odor of e.coli with pJBEI6409 plasmide
'by melanie' Transformation of competent cells 5mg1655) by electroporation (pJBEI6409) [1] We do some stock
Extraction of the Genomic DNA
by Juliette & Terry
From liquid culture made the EDIT !!!!
Polymerase chain reaction
by Sean & Pierre
for this PCR, five tubes of each of the following Bio-bricks were prepared.
BBa_K517003
| component | volume |
|---|---|
| H2O | 35.5μl |
| Phusion buffer 5X | 10μl |
| dNTPs | 2μl |
| iPS68bis | 1μl |
| iPS69 | 1μl |
| DNA | 1μl |
| Phusion enzyme | 0.5μl |
BBa_K762100
| component | volume |
|---|---|
| H2O | 35.5μl |
| Phusion buffer 5X | 10μl |
| dNTPs | 2μl |
| iPS66 | 1μl |
| iPS67 | 1μl |
| DNA | 1μl |
| Phusion enzyme | 0.5μl |
Tubes were placed in PCR machine with the following parameters.
| Cycle step | Temperature | Time | Cycle |
|---|---|---|---|
|
Initial denaturation |
98°C |
1 min |
1 |
| Denaturation | 98°C | 15 s | 25 - 30 |
| Annealing | 52°C | 25 s | 25 - 30 |
| Extension | 72°C | 45 s | 25-30 |
| Final extension | 72°C | 10 min | 1 |
| Final extension | 8°C | hold | 1 |
Members present:
- Instructors and advisors: Alice.
- Students: Eugene, Fabio, Hoang Vu, Juliette, Melanie, Pierre, Romain, Sean and Terry.
