Team:Paris/Notebook/Protocols
From 2008.igem.org
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* Run the whole samples (30 µL) in a '''0.8-1% agarose gel''' (a new one) | * Run the whole samples (30 µL) in a '''0.8-1% agarose gel''' (a new one) | ||
- | * Run at 50 | + | * Run at 50 V until halfway |
* 10 µL of ladder 1kb and 100 pb on every side | * 10 µL of ladder 1kb and 100 pb on every side | ||
* 30 µL of DNA + 6 µL of LB | * 30 µL of DNA + 6 µL of LB | ||
!!!Separate each band by an empty one!!! | !!!Separate each band by an empty one!!! | ||
- | |||
==Extraction== | ==Extraction== |
Revision as of 11:50, 13 August 2008
Culture of Stable strain with biobricks 2008
Glycerol Stocks
Minipreps (Kit Qiagen)
ElectrophoresisAn electrophoresis can be done to check if there is Product of Miniprep
Concentration of the MiniprepBy biophotometry
Check if the ratio 260/280 is over 1,6
Digestion
Migration after digestion for vectors
| Separate each band by an empty one | !
Extraction
Amplification of promoters=>To amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.
For each samples,
1 µl dNTP
LID : 105°C
Purification (Kit Promega)Gel Slice and PCR Product PreparationDissolving the Gel Slice
Processing PCR reactionsFor products above 40 pb
Binding of DNA
Washing
Elution
Quantification by electrophoresis
Ligation
TransformationUse of TOP10 chemically competent cells
PCR ScreeningUse of 8 clones of Ligation transformants for screening PCR
After, add
LID 105°C
Electrophoresis Purification of PCR
Sequencing |
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