Joyce
Biobricks were checked (by sequencing)
Transform: Digestion analysis: Sequencing:
PlasB: OK OK
RFP: OK OK
cILVA: OK OK
DT: OK OK
rev DT: OK OK
PBADaraC: OK OK
PcI: - -
GFP-LVA: - -
RBS-GFP-DT: OK OK
Plan: Digestion of RBS-GFP-DT with EcoRI and XbaI
PlasB and PBADaraC with EcoRI and SpeI
isolate both of gel and use also normal PBADaraC sample for ligation:
Digestion vector:
2μl FD buffer
0.5μl EcoRI
0.5μl XbaI
7μl MQ water
10μl mix+ 10μl sample
Digestion mix insert:
14μl FD buffer
3.5μl EcoRI
3.5μl SpeI
49μl MQ water
Digest for 45 min at 37 degrees
Clean up of the DNA with the PCR purification kit
Ligation:
7μl insert
3μl plasmid
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
7μl MQ water
Ligate for 2hours at room temperature and afterwards,ligate overnight
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