Team:Freiburg/Notebook/5 September

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green light receptor

transformation with reporter parts

Investigators:Julia

transformation with iGEM parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15016 BBa_I15016] which is B0032.ECFP
and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I15017 BBa_I15017 ]which is B0032.EYFP.
Tomorrow the green light promotor from PCR of 29_August should be inserted infront of the reporter proteins CFP and YFP.

blue light receptor

Miniprep

Investigators:Sophie
continue from: picking clones; 04.09.11

Testdigest showed no inserts

Ligation

Investigators: Sophie
again ligation as last time (02.08.11)

Lysis cassette

Troubleshooting of the modified Lysis genes K124017

Investigators:Theo

Minipreps of the first inoculations were applied on a 1% agarose gel without digesting them to see if they have an insert of the modified Lysis genes K124017. Nr1, 5 and 7 were sent for sequencing since the size seems to be about right.

• Note: S4 is the B0034 RBS (ribosome binding site), S15 is the non-modified K124017 Lysis Cassette without RBS

Precipitator

Digestion

Procedure

1. add H₂O (38μl-DNA )
2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
6. heat for 1-2 hours 37°C (6 hours if time)
7. heat for 20 minutes 80°C (inactivation of enzymes)
8. keep at 4°C if you cannot continue

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:

Restriction enzymes you need to cut the vector, insert1 and insert 2:

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.

Run a gel to verify if the part is cut out.

Miniprep

Investigators: Sophie
continue from experiment: Transformation; 03.09.11
testdigest showed no inserts

Trafo

Investigators. Sandra

Repeat of the trafo of:

1. pGEX vector (Amp. resistance)
2. pGEX vector (Amp. resistance)
3. pGEX vector (Amp. resistance)
4. pSB1C3
5. pET Duet vector (Amp. resistance)
6. pET Duet vector (Amp. resistance)
7. pET Duet vector (Amp. resistance)
8. pSB1C3
9. pSB1C3
10. pSB1C3