We ligated promoters into death vector, and transforned into Top 10. Our transformation worked, but it was hard to say with the result of yesterday if we had the good insert. Moreover, it is possible that we inverted Pupp and Pspac. So we are going to make a new single colony PCR with the primers of promoters (we used the same single colonies than yesterday).
We also want to PCR again all promoters (Pspac, Pxyl, Pupp and Ppac) from plasmids to have more.
Picture of the gel with the different promoters
promoter
colony
primers
name
Pspac
1
Pspac primers
A
Pspac
1
Pupp primers
B
Pspac
2
Pspac primers
C
Pspac
2
Pupp primers
D
Pupp
1
Pupp primers
E
Pupp
1
Pspac primers
F
Pupp
3
Pupp primers
G
Pupp
3
Pspac primers
H
- Single colony PCR : add 10μL of SDW, 5μL of MM, 1μL + 1μL of primers, 3μL of cells
- Gel
lane 3 : hyperladderI
lane 4 : Pspac (colony1) with Pspac primers
lane 5 : Pspac (colony1) with Pupp primers
lane 6 : Pspac (colony2) with Pspac primers
lane 7 : Pspac (colony2) with Pupp primers
lane 8 : Pupp (colony1) with Pupp primers
lane 9 : Pupp (colony1) with Pspac primers
lane 10 : Pupp (colony3) with Pupp primers
lane 11 : Pupp (colony3) with Pspac primers
lane 12 : HyperladderI
- Results
promoter
colony
primers
observation
Conclusion
Pspac
1
Pspac primers
nothing
it is in fact Pupp
Pspac
1
Pupp primers
band (about 280bp)
Pupp OK
Pspac
2
Pspac primers
nothing
it is in fact Pupp
Pspac
2
Pupp primers
band (about 280bp)
Pupp OK
Pupp
1
Pupp primers
nothing
it is in fact Pspac
Pupp
1
Pspac primers
band (about 180bp)
Pspac OK
Pupp
3
Pupp primers
nothing
it is in fact Pspac
Pupp
3
Pspac primers
band (about 180bp)
Pspac OK
Make some stock of our new biobricks
Pupp (colony1) into the death vector, transformed into TOP10
Pspac (colony1) into the death vector, transformed into TOP10
agrD (colony5) into the death vector, transformed into TOP10
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