"
Team:Groningen/Labwork/29 July 2013
From 2013.igem.org
| Line 44: | Line 44: | ||
Made a ligation for the knockout system of Des, with Des UP, Tet and Des Down. | Made a ligation for the knockout system of Des, with Des UP, Tet and Des Down. | ||
| + | <p> | ||
<h2>Chaline & Mirjam</h2> | <h2>Chaline & Mirjam</h2> | ||
Made a PCR reaction on the ligation of knockout Des, with an annealing temperature of 55°C and an expected size of 3200 bp. Gel examination revealed that only a smear appeared. | Made a PCR reaction on the ligation of knockout Des, with an annealing temperature of 55°C and an expected size of 3200 bp. Gel examination revealed that only a smear appeared. | ||
Revision as of 18:15, 29 July 2013
Mirjam
Run the samples of the colony PCR of BBa_K823823 combined with the LacI promoter made on 27-07 on gel (order 1-8).There are no bands around 1500 bp.
Run the purified PCR product of spec on gel.
The purified spec shows a nice band.
Run the PCR products of silk 2 on gel to do gel purification.
Only one of the tubes showed a band around the expected height.
Run the PCR products of silk 3 on gel to do gel purification
No bands appeared at the expected height.
Made a double restriction digestion with fast restriction digest for Pdes and CheY with respectively EcoRI and BamHI; and BamHI and PstI. Only a band appeared for Pdes.
Made a fast restriction digestion for RFP and eYFP and LacI+BBa_k823823. This time a control mechanism is used. So the fluorescent proteins are cut with EcoRI, PstI and EcoRI&PstI. As control also uncut eYFP is loaded on gel. It is noticed that the cut with EcoRI shows bands. When PstI is added, the bands appear.
So a new reaction digestion is made. This time using the EcoRI and the PstI from NEB. The restriction for LacI+BBa_k823823 is made and it works.
Chaline
Made a ligation for the knockout system of Des, with Des UP, Tet and Des Down.
Chaline & Mirjam
Made a PCR reaction on the ligation of knockout Des, with an annealing temperature of 55°C and an expected size of 3200 bp. Gel examination revealed that only a smear appeared.
Friso
Made a restriction digestion for Spec purified.
Claudio
Two GFP reporters are picked from the iGEM database: BBa_E0840 and BBa_E0240 (backbone: pSB1C3). Those are transformed into E. Coli DH5Α (Cm resistance).The plasmid containing mKate2 (red fluorescent protein into pGEM-T Easy vector) is inoculated in LB + Ampicillin resistance.
The Colony PCR which failed on Saturday is performed again on 4 colonies (the same which were also tested the previous time) changing the annealing temperature to 46°C.
The four samples are loaded on gel and bands are spotted around the expected height (~1500 bps).
The transformation worked.
|
|
|
|
 |
|
