IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/15: Difference between revisions
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- Plate out on Amp 100 (Neat) | - Plate out on Amp 100 (Neat) | ||
==Biobrick | ==Biobrick E0040 and I13522 (PA)== | ||
* PCR E0040 colonies 4 and 5 after miniprep of plasmid to ensure plasmid identity again | |||
* Protocol: SDW 7.5μL | |||
MasterMix 10μL | |||
VR and VF2 1x2μL | |||
DNA sample 0.5μL | |||
* Run on 1.2% SyBr E-gel | |||
* Lane 2 - 100bp ladder | |||
* Lane 3 - E0040 colony 4 | |||
* Lane 4 - E0040 colony 5 | |||
* Result: Bands at around 1000bp on both lanes 3 and 4 with a brighter band for lane 3. Correct band as E0040 VF-VR is 958bp | |||
* Minprep of plasmid E0040 from colony 4 is kept | |||
* Transform chemically compotent TOP10 with I13522 biobrick extract again | |||
* Transformation following standard protocols | |||
* TOP10 with I13522 plated neat and 1/10 on Amp. 100 plates and incubate at 37°C overnight | |||
* Will pick single colonies tomorrow for PCR analysis and into LB with Amp100 |
Revision as of 03:45, 28 October 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) Biobrick E0040 and I13522 (PA)
MasterMix 10μL VR and VF2 1x2μL DNA sample 0.5μL
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