Team:Freiburg/Notebook/6 September

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Commons

Miniprep

Investigators: Sandra

Yesterday LB was inoculated with strains (S14, S39-S44 etc.). Miniprep of strains.

Digestion: 2A-assembly

Investigators: Sandra

Digestion of:

• S14 (GFP)

with XbaI and PstI

• S39 (PR1)
• S40 (PR2)
• S41 (PR3)
• S42 (PR4)
• S43 (PR5)
• S44 (PR6)

with SpeI and PstI

incubation for 6 hours at 37°C.

green light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

blue light receptor

Trafo

Investigators: Sandra

Trafo of ligated parts of the 2A-assembly.

Sequence analysis

sequence analysis of our lovtap-Notgate was neagtive:-( So we´ll try the gibson assembly.

PCR

Investigators: Sophie PCR

PCR-Mixture for one Reaction:

For a 50 µl reaction use

Digestion with Dpn I and purification with PCR purification kit What program do you use?

First 20 cycles touchdown 69°C -0.4/cycle

next 10 cycles touchdown 72°C -0.2 per cycle

To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.

How did you label the PCR-Product, where is it stored and what do you do next?

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME

Lysis cassette

Troubleshooting of the modified Lysis genes K124017

Investigators:Theo

S4+S15 Nr1, 5 and 7 were sent for sequencing. Some problems arose and the sequences seemed to not be correct or only parts of them. So I decided to digest all of the minis and if an insert band (ie about 1300bp, since it is K124017 with RBS) was seen, to extract it from the gel and use it for subsequent ligation with the Promotor.

M66 which is (or SHOULD be -arrghh-) the alreadz modified Lysis genes K124017 was digested with EcoRI and PstI and two bands at around 1100 and 1400bp were seen, so both of them were extracted. I want to clone them on the pSB1C3 Vector and sequence that so that the part is also ready for the parts registry. More on that tommorow.

• Note: S4 is the B0034 RBS (ribosome binding site), S15 is the non-modified K124017 Lysis Cassette without RBS
  • Note 2: S4+S15 Nr1, 5 and 7 were digested with XbaI + PstI so that they can be one-step cloned with the temperature sensitive promotor (to be cut with SpeI and PstI and then treated with alcalic phosphatase as well as with the Qiagen PCR purification kit)

INSERT GEL EXTR KIT SOP HERE!!!!

Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME