Team:NRP-UEA-Norwich/Week10

From 2012.igem.org

(Difference between revisions)
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. As a backup, the running some of the running culture of the Comparator circuit 1 and 2 transformed cells were mini-prepped. After nanodropping, the concentration was found to be:
. As a backup, the running some of the running culture of the Comparator circuit 1 and 2 transformed cells were mini-prepped. After nanodropping, the concentration was found to be:
 +
C1-1a: 733.4 ng/µL
C1-1a: 733.4 ng/µL
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. Following this an overnight double restriction digest with ''Eco''R1 and ''Pst''1 was set up. These were set up using: 0.2µl  BSA, 2µl  Buffer H, 0.5µl  EcoR1 and 0.5µl  Pst1. As to the DNA, 1µg of DNA was added:
. Following this an overnight double restriction digest with ''Eco''R1 and ''Pst''1 was set up. These were set up using: 0.2µl  BSA, 2µl  Buffer H, 0.5µl  EcoR1 and 0.5µl  Pst1. As to the DNA, 1µg of DNA was added:
 +
C1-1a: 1.36 µL
C1-1a: 1.36 µL
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. M-B samples were digested with ''Spe''1 and ''Pst''1 to linearise the backbone to ligate a reporter (CFP, or RFP) into it. The small fragment that was released was discarded during gel purification. At the same time CFP and RFP were digested with ''Pst''1 and ''Xba''1 and then gel purified ready for ligation with M-B. The restriction digests involved 0.5µL of each enzyme, 2µL of Buffer B (''Spe''1 and ''Pst''1)and Buffer H (''Pst''1 and ''Xba''1), 0.2µL of BSA. Again 1µg of DNA was used. The gel used to separate the fragments was 1% w/v.  
. M-B samples were digested with ''Spe''1 and ''Pst''1 to linearise the backbone to ligate a reporter (CFP, or RFP) into it. The small fragment that was released was discarded during gel purification. At the same time CFP and RFP were digested with ''Pst''1 and ''Xba''1 and then gel purified ready for ligation with M-B. The restriction digests involved 0.5µL of each enzyme, 2µL of Buffer B (''Spe''1 and ''Pst''1)and Buffer H (''Pst''1 and ''Xba''1), 0.2µL of BSA. Again 1µg of DNA was used. The gel used to separate the fragments was 1% w/v.  
-
. Multiple ligations were set up. MB2, MB3,MB5,MB9,MB10 were set up to ligate with both RFP and CFP. Comparator circuit 1 and 2 (all samples) were set up to be ligated in the iGEM backbone pSB1C3. These were all left overnight.
+
. Multiple ligations were set up: MB2, MB3,MB5,MB9,MB10 were set up to ligate with both RFP and CFP and Comparator circuit 1 and 2 (all samples) were set up to be ligated in the iGEM backbone pSB1C3. These were all left overnight.
==Day 2 (11/09/12)==
==Day 2 (11/09/12)==

Revision as of 11:12, 12 September 2012

Header1NewGreen.png

NRP UEA iGEM 2012

 

Welcome to the NRP UEA iGEM 2012 Wiki Lab Book

Please choose the relevant link to access our diary of that week!

Week Zero | Week One | Week Two | Week Three | Week Four | Week Five | Week Six | Week Seven | Week Eight | Week Nine | Week Ten | Week Eleven | Lab Protocols | Experiments

Contents

Week 10

Day 1 (10/09/12)

. As a backup, the running some of the running culture of the Comparator circuit 1 and 2 transformed cells were mini-prepped. After nanodropping, the concentration was found to be:

C1-1a: 733.4 ng/µL

C1-2a: 1192.6ng/µL

C1-3a: 1596.9 ng/µL

C1-4a: 854.8 ng/µL

C2-1a: 896 ng/µL

C2-2a: 712.5 ng/µL

C2-3a: 1196.7 ng/µL

C2-4a: 811.9 ng/µL

. Following this an overnight double restriction digest with EcoR1 and Pst1 was set up. These were set up using: 0.2µl BSA, 2µl Buffer H, 0.5µl EcoR1 and 0.5µl Pst1. As to the DNA, 1µg of DNA was added:

C1-1a: 1.36 µL

C1-2a: 0.84 µL

C1-3a: 0.62 µL

C1-4a: 1.17 µL

C2-1a: 1.12 µL

C2-2a: 1.40 µL

C2-3a: 0.84 µL

C2-4a: 1.23 µL

. Following a low concentration reading on Friday, B-M was miniprepped again. However, this produced no DNA when nanodropped. Therefore, B-M was reinoculated into culture.

. M-B samples were digested with Spe1 and Pst1 to linearise the backbone to ligate a reporter (CFP, or RFP) into it. The small fragment that was released was discarded during gel purification. At the same time CFP and RFP were digested with Pst1 and Xba1 and then gel purified ready for ligation with M-B. The restriction digests involved 0.5µL of each enzyme, 2µL of Buffer B (Spe1 and Pst1)and Buffer H (Pst1 and Xba1), 0.2µL of BSA. Again 1µg of DNA was used. The gel used to separate the fragments was 1% w/v.

. Multiple ligations were set up: MB2, MB3,MB5,MB9,MB10 were set up to ligate with both RFP and CFP and Comparator circuit 1 and 2 (all samples) were set up to be ligated in the iGEM backbone pSB1C3. These were all left overnight.

Day 2 (11/09/12)

Day 3 (12/09/12)

Day 4 (13/09/12)

Day 5 (14/09/12)

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