Team:PKU Beijing/Notebook/AND Gate 1/Input/TetR

Notebook > AND Gate 1 > Input > aTc Sensor

2009.7.4
Back in the Lab. Tranformation(Parts listed below)

2009.7.5
Pick colonies of the transformation and shake in the incubator.

2009.7.6
MiniPrep tetR standard parts plasmid.(BBa_C0040) Get 6 standard rbs plasmids From ShenShan.

21:40 Digest rbs

Digest tetR

2009.7.7
01:52 GEL to assess the digestion CIAP the 6 rbs vectors. Add to the digestion product 5ul CIAP Buffer and 1ul CIAP

Run a GEL to recycle the insert of tetR. The Hole is too large, the band is missing. (Never use the largest Cone for 20ul recycle any more)

03:24 Digest the tetR plasmid again.

2009.7.8
The Primer(Made of Standard Prefix and Suffix arrived). Add ddH2O to each tube.

PCR tetR plasmid (MasterMix) to see whether the primers work. PCR protocol. (the Band is too narrow which means that the PCR is not done very well)

Try Colony PCR No Signal

Go to Lingli’s Lab, do gradient PCR. 52, 53, 54, 55, 56, 57, 62, 64

All of the temps works well.

It turns out that the thermocycler in our lab has no hot cap. So the liquid is on the tube cap when exposed to 94 centigrade.

2009.7.15
22:16 Help Wushuke with his PCR. The lacI1-1, lacI2-2, tetR2-2, tetR3-2, tetR3-1.

2009.7.16
MiniPrep 1-18P and 2-4O plasmid Enzyme Digestion of 1-18P and 2-4O plasmid Several Trials and failures, 2-4O can not be digested normally, at least be TaKaRa Enzymes. Check up the parts and found BBa_J09855 Constitutive LuxR with pLuxR 1-9H pSB1A2