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User:University of Washington/25 August 2008
From 2008.igem.org
Conjugation BioBricks (Scott)
·PCR TrbA gene
·grow TrbC gene for minipreps and sequencing
·purify KorA gene for ligation tomorrow
LuxR from AraC and TetR(Faifan)
- got no growth from second time inoculating the transformation of ligated DNA(promoter + GFP/LuxR + pSB3K3)
- grew overnight I0462 from glycerol stock x 3 with Amp
- started cloning protocol from Ingrid (after digestion, will treat with CIP to remove phosphate from 5' end so the DNA won't re-ligate to itself.)
- Restriction Digest: mix
| dH2O | Buffer | BSA | DNA | XbaI | SpeI | PstI | |
|---|---|---|---|---|---|---|---|
| promo SP | 10.5 | NEB2, 5 | 0.5 | 30 | - | 2 | 2 |
| promo S | 15.6 | NEB2, 2 | 0.2 | 2 | - | 0.2 | - |
| promo P | 15.6 | NEB3, 2 | 0.2 | 2 | - | - | 0.2 |
| promo no enz | 15.8 | NEB2, 2 | 0.2 | 2 | - | - | - |
| GFP | 10.5 | NEB3, 5 | 0.5 | 30 | 2 | - | 2 |
| LuxR | 10.5 | NEB3, 5 | 0.5 | 30 | 2 | - | 2 |
- incubated 37 degree Celcius for 2 hrs
- 20 mins denature at 80 degree Celcius
- stored digestion product in -20
- ran gel on promo SP, S, P, and no enz: used 3 ul DNA, 2 ul dye
- Bands of promo SP, S, P ran differently from no enz and were around at 3 kb as expected. However, there seemed to be light bands at higher than 10 kb ladder in SP lane which could indicate that not all plasmid were digested.
MG1655Z1(Faifan)
-none grew in four antibiotic plates ==> lost pCS26 with Kan, pACYC184 with Cam/Tet??, and some plasmid with Amp.. likely??
-grew overnight from the culture from Tsy culture#3 plate.
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