Team:Paris/August 14
From 2008.igem.org
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====Washing of the digestions==== | ====Washing of the digestions==== | ||
We washed the DNA following the [[Team:Paris/Notebook/Protocols#Purification_.28Kit_Promega.29|standard protocol]]. | We washed the DNA following the [[Team:Paris/Notebook/Protocols#Purification_.28Kit_Promega.29|standard protocol]]. | ||
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+ | ===Ligation=== |
Revision as of 12:24, 15 August 2008
Contents |
Results of the PCR of the wonderful promoter of fliL
Electrophoresis settings :
- Gel 1.5% agar
- Ladder 100 bp
- Volume of template : 3 µL
Name | Promotor | Well | Expected size | Measured size |
PCR_135 | pfliL | 2 | 197 bp | |
PCR_135' | Negative Control | 3 | 0 bp |
Remarks: We observe that the bands are curved, we suppose that the wells were not very clean. The size of fliL is good, we will digest it and ligate it today.
Digestion and Ligation of the wonderful promoter of fliL
As the primer used to amplify the promoter of fliL had only two nucleotides after the restriction sites, we tried the two digestions possible : EcoRI + SpeI and XbaI + PstI.
Digestion
Protocol :
Name | Genes | Water | DNA | Buffer n°2 10X | BSA 100X | Enz 1 | Enz 2 |
D149 | fliL | 23 µL | 2 µl | 3 µl | 0.30µl | EcoRI | SpeI |
D150 | fliL | 23 µL | 2 µl | 3 µl | 0.30µl | XbaI | PstI |
D137 | pSB3K3 | 23 µL | 2 µl | 3 µl | 0.30µl | EcoRI | SpeI |
D152 | pSB3K3 | 23 µL | 2 µl | 3 µl | 0.30µl | XbaI | PstI |
- Incubate 2h30 at 37°C
- 20 min at 65°C to denaturate the enzymes.
Washing of the digestions
We washed the DNA following the standard protocol.