Team:Goettingen/notebook wetlab/biobricks

July

- Used BioBricks from library:
- Tick with a tip a hole in plate and resolve the pellet in 10µl sterile water, red color was visible, pipeting up and down, wait 5 minutes and put 10 µl into a PCR cup and freeze it
- 1 µl is transformed into DH5α → plated on Cm or kan plates
- Transformation worked, pick colonies for o/n in LB + Cm to isolate BB-plasmids
- Isolation of plasmids

Agarose gel (1%):

August

→After several tries to construct our BioBricks on the described way we changed to seamless cloning technique

- PCRs for seamless cloning:

Agarose gels (1%):
- Purification of the products and measurement of the concentration of each product.
- Amplification of Leu2, Trp1 and 2-micron with 2 sets of primers to create mutated genes that loose restriction sites:

1. PCR to create for each gene 2 fragments that contain 15 bp overhangs to the other one.
2. Use fusion PCR approach to fuse genes with silent mution together.
3. Gel extract the rightly fused PCR product and use it in a normal PCR for amplification.
- Purification and sequencing of this three samples

September

- results sequencing:

Trp1 AGGCCGCAGAATGTGCTCTGGATTCCGATGCTGACTTGCT
Trp1*AGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCT

Leu2 TGGTCCTAAATGGGGTACCGGTAGTGTTAGACCTGAACAA
Leu2*TGGTCCTAAATGGGGTACAGGTAGTGTTAGACCTGAACAA

2-micron GAAGTTCCTATACTTTCTAG****AGAATAGGAACTTCGGAATAGGAACTTC
2-micron*GAAGTTCCTATACTTTCTAGCTAGAGAATAGGAACTTCGGAATAGGAACTTC

⇒ silent mutations delete the restriction sides

- Prepare seamless mixture (see protocol and formula) with I – VI
- Transformation of the whole reaction sample into DH5α.
- Grown colonies on plates → pick several colonies and grow over night.
- Colony PCR with primer on backbone and primer in insert
- plasmid isolation of positives

- Amplification of construct via PCR
- Gel extraction & Purification & seamless cloning with A-F
- Transformation into DH5α
- Pick colonies and isolate plasmids from overnight culture
- colony PCR to verify construct
- restriction of the two different plasmids and the fusion product of 2-micron ligation over night of the 2-micron behind restriction site in plasmid backbones
- Transformation into DH5α.
- Pick several colonies into 4 mL LB +Cm for overnight cultures.
- Colony PCR and isolation of plasmids.
- Chose positives for further work and shipping to the registry