IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/29

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(Single colony PCR to check our transformation)
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Revision as of 06:50, 5 September 2008



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Results of transformation with our ligation products

  • Everything grew! Better efficiency with electrop. than with chemical protocol

Single colony PCR to check our transformation

Transformed products number of picked colonies from chemical transformation number of picked colonies from electrop. transformation (neat) number of picked colonies from electrop. transformation (1:10)
Pupp 2 2 0
Pspac 2 2 0
RBS S 3 0 2
RBS W 3 0 2
agrD 3 2 0

- Single colony PCR : 13μL of SDW+cells, 5μL of Master Mix, 1μL of VF and 1μL of VR (and plate each single colony)

- Load a gel (1.3% agarose) : 5μL of PCR products + 1μL of dye (only 1μL of 100b ladder)

In the death plasmid, the VF-VR size is about 280b.

Transformed products size of the product (with cutting sites) expected size after PCR (about)
Pupp 255 480
Pspac 125 350
RBS S 56 280
RBS W 56 280
agrD 200 430
  • Result : nothing, even no ladder, problem with the gel!

- run again on a e-gel

  • Result : nothing, just the primers! Problem with our PCR

Stock of ECE112

- Bulk up ECE112 (Amp100) rfom 10mL LB (28/08), take 1mL and add to 100mL of LB in big flask

- Grow overnight in 37°C incubator with vigorous shaking

- Aliquot in 10mL tubes, pellet and freeze cell pellets

Transformation of glycerol stock

  • Strian : IA751

- TTransfer into Eppendorf tubes and spin down for 30min

-Remove glycerol and add medium B

- Add ECE112 and ECE153, and one control tube

- incubate for 30min in 37°C

- Plate out ECE112 (Cm5), ECE153 (Spc50), DNA less (blank, Cm5, Spc50)

Plate biobricks from MIT

  • E0840
  • B0014
  • I712007
  • C0012
  • B0015
  • C0061
  • R0063


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