green light receptor
second quickchange of CcaS
Investigators:Julia
| Name: Julia
| Date: 22.08.11
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| Project Name: second quickchange of CcaS
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PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl
| H20
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| 10µl
| 5x Phusion Buffer
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| 2.5µl
| Primer fw
| CGGATCATCAATGGGCTCC (P64)
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| 2.5µl
| Primer dw
| GGTCCGCGCATTCTCTATGTCAATGAAGCAT (P65)
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| 1µl
| dNTPs
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| 1µl
| DNA-Template
| qS50 and qS51 (5ng/ µl)
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| 0.5 µl
| Phusion (add in the end)
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What program do you use?
| Temperature
| time (min)
| No. of cycles
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| 95
| 5:00
| 1
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| 95
| 0:15
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| 60
| 0:15
| 20
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| 72
| 1:15
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| 72
| 5:00
| 1
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| 4
| 60:00:00
| 1
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After PCR: Digestion with DpnI in order to get of the old plasmids :
5 µl NEB buffer 4
5 µl BSA (10x)
2 µl DpnI
38 µl of PCR Product
Incubation for 1,5h, deactivation at 80°C for 20 min.
Transformation with 10 µl of the digested PCR product. Cells were plated out on Kanamycin plates( at 23:00 o’clock ).
blue light receptor
Sequencing
Investigators: Sandra
The sequencing showed that the 3A-assembly did not work.
We will send the LovTap PCR product to get sequenced. May be the PCR did not work.
PCR
Investigators: Sandra
PCR of Not-Gate.
P 74:
P 24:
PCR product was loaded onto a gel. Expected lane: 900bp
PCR worked and we got Not-Gate with NEHI and PstI.
Cloning
Investigators: Sophie
new 3A assembly with Sandra's NOT-gate-PCR-product, our LOV-Tap-pcr-product (both mutated with NEH1 restriction sites) and an amp-vector
The digested samples were loaded to a gel. They showed clear bands in the right size. Only the band of the vector was not intense enough, so the vector has to be digested again.
red light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
Lysis cassette
NAME OF YOUR EXPERIMENT
Investigators:NAME
Precipitator
Transformation
Investigators: Rüdiger, Sandra
Transformation of:
61 a,b
62 a,b
and 63 a,b.
Vector:pSB1C3
Incubation over night.
Cloning for GFP-pbd with IPTG-inucible promoter
Investigator: Sophie
as cloning from 18.08.11 did not work I try it again with longer digestion times.
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