IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/15: Difference between revisions
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- Maybe we did not add enough DNA, or there is another problem. We will e mail the guy from Bacillus center before trying again, because we don't have enough DNA. | - Maybe we did not add enough DNA, or there is another problem. We will e mail the guy from Bacillus center before trying again, because we don't have enough DNA. | ||
== | ==Plasmid miniprep ECE 151 (x2), ECE 153, ECE 166== | ||
*'''Nanodrop results''' : | *'''Nanodrop results''' : | ||
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==Xylose Induction with ECE 153== | |||
- 4 Tubes | - 4 Tubes | ||
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==Glycerol Stock Transformation== | |||
- Spin down cells (competent) from 5/8 and 13/8 stocks | - Spin down cells (competent) from 5/8 and 13/8 stocks | ||
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==Transforming ECE 188, 189, 190 (3rd try)== | |||
- 2 tubes of chemically competent top 10 | - 2 tubes of chemically competent top 10 | ||
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==PCRing out promoters== | |||
- 4 different promoters : Pxyl, Pspc, Ppac, Pupp | - 4 different promoters : Pxyl, Pspc, Ppac, Pupp | ||
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==Digest and Ligate Agr A and C to pSB4C5== | |||
Previous PCR products of Agr A and C were digested using Fermentas Fast Digest - EcoRI and PstI according to their protocol except a longer time was given to digest. | Previous PCR products of Agr A and C were digested using Fermentas Fast Digest - EcoRI and PstI according to their protocol except a longer time was given to digest. | ||
Revision as of 08:40, 5 September 2008
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Transformation ECE188, 189, 190 into E.coli- Results : only our control plate grew!! - Maybe we did not add enough DNA, or there is another problem. We will e mail the guy from Bacillus center before trying again, because we don't have enough DNA. Plasmid miniprep ECE 151 (x2), ECE 153, ECE 166
Xylose Induction with ECE 153- 4 Tubes - No fluorescence! We have to think about the transformation of integration vectors into Bacillus.
Glycerol Stock Transformation- Spin down cells (competent) from 5/8 and 13/8 stocks - Remove liquid, resuspend in Medium B - Incubate for 60 mins at 37°C - Add ECE 166 into tubes - Incubate for 30 mins at 37°C - Plate out on CM 5 plates - 4 plates
Transforming ECE 188, 189, 190 (3rd try)- 2 tubes of chemically competent top 10 - Spin down, remove liquid - Resuspend cells in 100μL of CaCl2 50mM - 4 tubes with 50μL of cells
- Ice for 30 mins - 42°C for 2 mins - Ice for 2 mins - Incubate at 37°C for 2 hours - Plate out on Amp 100 (Neat)
PCRing out promoters- 4 different promoters : Pxyl, Pspc, Ppac, Pupp - Make master mix enough for 4RXNs
Digest and Ligate Agr A and C to pSB4C5Previous PCR products of Agr A and C were digested using Fermentas Fast Digest - EcoRI and PstI according to their protocol except a longer time was given to digest. 3uL of AgrA was used and 4.4 uL of AgrC was used and incubated for 40minutes. 10uL totaling to 1ug of pSB4C5 plasmid was also digested and incubated for 10 minutes. The digestion was visualized on a 0.8% Agarose gel and produced expected sizes: Plasmid: approx 3bk Agr A: approx 700bp Agr C: approx 1300bp
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