Team:Cambridge/LabBook/Week9

From 2010.igem.org

(Difference between revisions)
Line 118: Line 118:
**Elongation: 15s @ 72°C
**Elongation: 15s @ 72°C
*30 cycles
*30 cycles
-
**Denaturation: 10s @ 98
+
**Denaturation: 10s @ 98°C
-
**Annealing: 20s @ 50
+
**Annealing: 20s @ 50°C
-
**Elongation: 15s @ 72
+
**Elongation: 15s @ 72°C
-
*End and hold at 10
+
*End and hold at 10°C
*We then ran result on a gel with SyberSafe at 6x lodaing Dye and Hyperladder IV
*We then ran result on a gel with SyberSafe at 6x lodaing Dye and Hyperladder IV
 +
 +
 +
{|class="wikitable"
 +
|-
 +
|Lane: 1
 +
|2
 +
|3
 +
|4
 +
|5
 +
|-
 +
|Ladder
 +
|Tube 1
 +
|Tube 2
 +
|Tube 3
 +
|Negative Control
 +
|}
 +
 +
Failed: Something obtained in control...
 +
 +
Nothing in other lanes
 +
 +
We repeated without touchdown PCR:
 +
*Denaturation 5min @ 98°C
 +
*35 Cycles
 +
**Denaturation: 12s @ 98°C
 +
**Annealing: 20s @ 58.5°C
 +
**Elongation: 15s @ 72°C
 +
*Hold at 10°C
 +
 +
Failed again: Product in negative control observed again...
 +
 +
==Tuesday==
 +
===91. Expt: Further testing===
µl°C
µl°C

Revision as of 14:39, 28 September 2010

Week 9: Monday 6th September - Sunday 12th September

Contents

Monday

86. Expt: Extract CDABEG from pJS555

  • Using 2 primers
    • prefix start of O
    • suffix end of G
  • Hope that it will glow in its own right without luxR+I
  • Need to put under a new promoter

87. Expt: Gibson transformation (cont. from p.70)

3 colonies on one plate were not pink. These were streaked out on new Chl plated and put into liquid cultures along with 2 of the red colonies.

Next check for the right sized fragment with colony PCR and try to induce with arabinose.

88. Expt: Plate Reader G28

Well Arabinose conc.
A1 0 x1,9
A2 1µM
A3 5µM
A4 10µM
A5 100µM
A6 Blank
A7 pSB1C3
A8 Blank x8,16

Reads every 10 mins

89. Expt: Extracting YFP, CFP, Luc tetR, G28 via miniprep (Paul)

Nanodrop readings (ng/µl)
YFP 33.3, 55.4
CFP 17.9, 20.4
Luc tetR 33.7
G28 125.3, 96.9
G28? 17.9

90. Expt: Colony PCR to extract the thioesterase gene from E. coli K12 (Paul)

We used TOP10 cells, a substrain of DH10B, which is a substrain of K12.

We did a colony PCR to isolate the gene:

3 replicates with: 1 negative control:
2x Phusion Mastermix 10µl 10µl
Template DNA 1µl 0
Primer 1 1µl 1µl
Primer 2 1µl 1µl
Nuclease-free H20 7µl 8µl

The negative control was to check for primer/dimer and contamination.

PCR protocol:

  • Initial denaturation: 5min @ 98°C
  • Touchdown: 16 cycles
    • Denaturation: 10s @ 98°C
    • Annealing: 20s @ 65°C to 57°C
    • Elongation: 15s @ 72°C
  • 30 cycles
    • Denaturation: 10s @ 98°C
    • Annealing: 20s @ 50°C
    • Elongation: 15s @ 72°C
  • End and hold at 10°C
  • We then ran result on a gel with SyberSafe at 6x lodaing Dye and Hyperladder IV


Lane: 1 2 3 4 5
Ladder Tube 1 Tube 2 Tube 3 Negative Control

Failed: Something obtained in control...

Nothing in other lanes

We repeated without touchdown PCR:

  • Denaturation 5min @ 98°C
  • 35 Cycles
    • Denaturation: 12s @ 98°C
    • Annealing: 20s @ 58.5°C
    • Elongation: 15s @ 72°C
  • Hold at 10°C

Failed again: Product in negative control observed again...

Tuesday

91. Expt: Further testing

µl°C

Retrieved from "http://2010.igem.org/Team:Cambridge/LabBook/Week9"