IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/08/02: Difference between revisions
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===Gel extraction=== | ===Gel extraction=== | ||
Gal4 | Gal4 spe bam 8/2 and EcR bam xba 8/2 gel extracted and purified. | ||
Gal4 on the left, EcR on the right | |||
[[Image:Gal4_Ecr_digest_gel_ex_8-2.jpg]] | [[Image:Gal4_Ecr_digest_gel_ex_8-2.jpg]] | ||
===Nanodrop of Gal4 and EcR Gel Extractions=== | |||
{| border="3" style="margin-left: 3em;" | |||
|- | |||
! Plasmid !! Quantity (ng/μL) !! 260/280 | |||
|- | |||
! Gal4 (1) | |||
| 1.4 || -3.18 | |||
|- | |||
! Gal4 (2) | |||
| 7.2 || 4.59 | |||
|- | |||
! EcR (1) | |||
| 5.3 || 2.52 | |||
|- | |||
! EcR (2) | |||
| 5.6 || 2.12 | |||
|} | |||
===VP16 PCR 2=== | |||
60μL reaction: | |||
*1.5 μL pfx polymerase | |||
*3μL DNTPs | |||
*18μL enhancer buffer | |||
*1.5μL MgSO<sub>4</sub> | |||
*3μL 1/10 dilution VP16.Hind.Fwd | |||
*3μL 1/10 dilution VP16.Rev | |||
*3μL 1/10 dilution of VP16 (1) | |||
*12μL amp buffer | |||
*5μL DH<sub>2</sub>O | |||
using program: | |||
1=94°C for 2:00 | |||
2=94°C for :15 | |||
3=68°C for :30 | |||
4=Goto 2, 30 times | |||
5=4.0°C forever | |||
6=end | |||
No PCR product is visible | |||
[[Image:VP16PCR.2_blank_8-2.jpg]] | |||
===VP16 PCR.3=== | |||
60μL reaction: | |||
*1.5 μL pfx polymerase | |||
*3μL DNTPs | |||
*18μL enhancer buffer | |||
*1.5μL MgSO<sub>4</sub> | |||
*3μL 1/10 dilution VP16.Hind.Fwd | |||
*3μL 1/10 dilution VP16.Rev | |||
*3μL 1/10 dilution of VP16 (1) | |||
*12μL amp buffer | |||
*5μL DH<sub>2</sub>O | |||
Using modified program: | |||
1=94°C for 2:00 | |||
2=94°C for :15 | |||
3=60°C for :30 | |||
4=70°C for :45 | |||
5=Goto 2, 30 times | |||
6=4.0°C forever | |||
7=end | |||
(see tomorrow's entry for gel image) | |||
===Ligations=== | |||
Gal4 into EcR | |||
Barnase into B15 | |||
LTP into B11 (positive control from team allergy) | |||
Gal4 | |||
*3μL Gal4 spe bam gel pur. (2) 8/2 | |||
*9μL EcR xba bam gel pur. (2) 8/2 | |||
*2μL 10x T4 ligase buffer | |||
*5μL DH<sub>2</sub>O | |||
*1μL T4 ligase | |||
EcR control | |||
*9μL EcR xba bam gel pur. (2) 8/2 | |||
*2μL 10x T4 ligase buffer | |||
*8μL DH<sub>2</sub>O | |||
*1μL T4 ligase | |||
Barnase | |||
*1.5μL Barnase digest gel pur. (1) 7/29 | |||
*3μL B15 spe pst phosphorylated clean up | |||
*2μL 10x T4 ligase buffer | |||
*12.5μL DH<sub>2</sub>O | |||
*1μL T4 ligase | |||
B15 control | |||
*3μL B15 spe pst phosphorylated clean up | |||
*2μL 10x T4 ligase buffer | |||
*14μL DH<sub>2</sub>O | |||
*1μL T4 ligase | |||
LTP | |||
*2μL LTP | |||
*1μL B11 backbone | |||
*2μL 10x T4 ligase buffer | |||
*16μL DH<sub>2</sub>O (should have been 14μL, the 16 was accidental) | |||
*1μL T4 ligase | |||
B11 control | |||
*1μL B11 backbone | |||
*2μL 10x T4 ligase buffer | |||
*16μL DH<sub>2</sub>O (should have been 14μL, the 16 was accidental) | |||
*1μL T4 ligase | |||
Allowed tubes to sit at room temp for an hour, proceeded to transformation | |||
==Team Flavor== | |||
===DTL of Mira/Brazz+StrepII+Stop into V24=== | |||
* Digested 1 microgram of DNA for each insert and for V24 backbone. | |||
** Digested inserts and backbone with NotI/SpeI | |||
* Gel extracted and purified bands indicated in image below | |||
* Ligated insert into 50ng of V24 backbone with a 3:1 insert to backbone ratio | |||
* Transformed and plated on LB+AMP plates and left in 37°C incubator overnight | |||
===DTL of Wintergreen pathway=== | |||
* Digested 400ng of J45004, 20ng of J45017, and 1 microgram of B21 (for v0120 backbone) | |||
** Digested J45004, J45017, and B21 BB with XbaI/PstI | |||
* PCR purified digested inserts | |||
* Gel extracted and purified B21 BB (see gel image below) | |||
* Ligated inserts into 50ng of B21 v0120 backbone with a 3:1 insert to backbone ratio | |||
** Transformed and plated on LB+AMP plates and left in 37°C incubator overnight | |||
===Gel Purification=== | |||
[[Image:Mbv24b21digest.jpg||240px]] | |||
# Ladder | |||
# Mira N SS | |||
# Mira C SS | |||
# Brazz N SS | |||
#Brazz C SS | |||
# V24 N/S | |||
# B21 X/P | |||
# Ladder | |||
*Gel bands indicated were cut and purified from the gel. | |||
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Revision as of 13:43, 7 September 2010
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Team FenceGel extractionGal4 spe bam 8/2 and EcR bam xba 8/2 gel extracted and purified. Gal4 on the left, EcR on the right Nanodrop of Gal4 and EcR Gel Extractions
VP16 PCR 260μL reaction:
using program: 1=94°C for 2:00 2=94°C for :15 3=68°C for :30 4=Goto 2, 30 times 5=4.0°C forever 6=end
VP16 PCR.360μL reaction:
Using modified program: 1=94°C for 2:00 2=94°C for :15 3=60°C for :30 4=70°C for :45 5=Goto 2, 30 times 6=4.0°C forever 7=end (see tomorrow's entry for gel image) LigationsGal4 into EcR Barnase into B15 LTP into B11 (positive control from team allergy) Gal4
EcR control
Barnase
B15 control
LTP
B11 control
Allowed tubes to sit at room temp for an hour, proceeded to transformation Team FlavorDTL of Mira/Brazz+StrepII+Stop into V24
DTL of Wintergreen pathway
Gel Purification
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