"
Team:Paris Saclay/Notebook/August/20
From 2014.igem.org
(→Migration of pPS1 digested by PacI for the purification) |
(→Digestion of CAD and PMCS5 at 37°C) |
||
| Line 214: | Line 214: | ||
After the migration, we want to purify the band corresponding of pPS1 digested by Pac1. While exposing the gel to UV, we will cut the band and save it inside an Eppendorf (weight = 1.080g) , before the purification | After the migration, we want to purify the band corresponding of pPS1 digested by Pac1. While exposing the gel to UV, we will cut the band and save it inside an Eppendorf (weight = 1.080g) , before the purification | ||
| - | === Digestion of CAD and PMCS5 at 37°C === | + | ====Digestion of CAD and PMCS5 at 37°C==== |
''by Eugene'' | ''by Eugene'' | ||
Revision as of 18:14, 22 August 2014
Contents |
Wednesday 20th August
Lab work
Petri dishes
by Laetitia
| Antibiotic | Concentration |
|---|---|
| XGal | 80μg/ml |
| IPTG | 2*10-3 |
| Ampicillin | 10-3 |
B - Construction of the fusion protein (color)
PCR
| components | volumes |
|---|---|
| DNA | .5µl |
| GoTaq buffer 5X | 10μl |
| DMSO | 1.5µl |
| H2O | 22.75µl |
| MgCl2 | 4µl |
| dNTP | 1µl |
| iPS83 | 5µl |
| iPS84 | 5µl |
| GoTaq polymerase | .25µl |
| step | temperature (°C) | time (min) |
|---|---|---|
| 1 | 95 | 2 |
| 2 | 95 | .5 |
| 3 | 58 | .5 |
| 4 | 72 | 1.75 |
| 5 | 72 | 5 |
Cloning in pGEMTesay
by Sean
D- Lemon scent
Bacterial culture
by Laetitia
Here are the results of the bacterial culture of yesterday
Bacteria with the pGEMTeasy without the DNA insert are blue and the bacteria with the pGEMTeasy and the DNA insert (PS, GS or LS gene) are white
Then, we have chosen 3 white colonies for each DNA insert and we have launched 3 corresponding liquid cultures :
- 5mL LB + ampiciline (1/1000e)
- 1 white colony
The 9 cultures are incubated at 37°C
by Melanie
Preculture 20ml of DH5a pPSI (apra 1/2000)
CAD PCR
Today we received our synthetic gene CAD (cinnamyl alcohol deshydrogenase) :)
so we do a PCR (with GoTaq):
| components | volumes |
|---|---|
| DNA of p cola | "very small amount" |
| GoTaq buffer 5X | 10μl |
| DMSO | 1.5µl |
| H2O | 23.25µl |
| MgCl2 | 4µl |
| dNTP | 1µl |
| iPS79 | 5µl |
| iPS80 | 5µl |
| GoTaq polymerase | .25µl |
| step | temperature (°C) | time (min) |
|---|---|---|
| 1 | 95 | 2 |
| 2 | 95 | .5 |
| 3 | 58 | .5 |
| 4 | 72 | 1.75 |
| 5 | 72 | 5 |
CAD cloning in pGEMTeasy
by Sean
Electrophoresis of pPSI digested by PacI
by Eugene
We made an electrophoresis of pPSI to verify if the digestion made 19th August has worked. It is useful before starting the purification of the plasmid.
- non-digested pPSI 3µl
- digested pPSI 3µl
- ladder 10µl
Migration of pPS1 digested by PacI for the purification
by Huang vu and Laetitia
pPS1 previously digested by PacI is filled inside an agarose gel :
-1g agarose
-1mL TAE
-2 µL BET
The totality of the sample is filled (around 38µL) and we filled 20µl of ladder. Migration 100V
After the migration, we want to purify the band corresponding of pPS1 digested by Pac1. While exposing the gel to UV, we will cut the band and save it inside an Eppendorf (weight = 1.080g) , before the purification
Digestion of CAD and PMCS5 at 37°C
by Eugene
| components | volumes |
|---|---|
| CAD | 5 μl |
| 10x buffer fastDigest | 1 μl |
| PacI | 0.5 µl |
| H2O | 3.5 µl |
| components | volumes |
|---|---|
| PMCS5 | 15 μl |
| 10x buffer fastDigest | 2 μl |
| PacI | 0.5 µl |
| H2O | 2.5 µl |
