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From 2013.igem.org

Contents 1 Results from previous day 2 Incubation of bacteria from the plates of previous day for mini prep 3 Sending pGEM-T vector with Tod insert clones to sequencing by PNACL 4 Digestion of new fusion PCR Tod genes

Results from previous day

• Background control plate had growth, which was not expected. This suggests that pSB1C3 backbone religated.
• It was also thought that the Tod genes used were not from the PCR fusion experiment.
• Same experiment done again, with a new set of Tod genes from a PCR fusion.
• For digestion of backbone, already digested backbone from 01/08/2013 was used, which was digested with EcoRI and PstI.

Incubation of bacteria from the plates of previous day for mini prep

• 12 single colonies were picked from each plate and put into 15ml of broth
• 36 tubes were left incubating overnight at 37C

Sending pGEM-T vector with Tod insert clones to sequencing by PNACL

Digestion of new fusion PCR Tod genes

• Digestion with EcorI-HF and PstI-HF using Cutsmart buffer
• 500ng of DNA was digested from the following Tod DNA concentrations:

Sample	Concentration ng/ul	260/280	260/230
Tod X	49.5	1.89	1.99
Tod F	33.9	1.98	1.68
Tob B	37.4	1.82	1.70

• Volumes for double digest:

Sample	DNA	Buffer	Water	EcorI-HF	PstI-HF
Tod X	10.1ul	3ul	15.9	0.5	0.5
Tod F	13.4ul	3ul	12.6	0.5	0.5
Tob B	14.7ul	3ul	11.3	0.5	0.5