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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 5 Ligation of Tod genes and pSB1C3 backbone 6 Transformation of TOD genes ligations with the digested pSB1C3 using the pGEM-T Vector System I-A3600 (changes were made to the protocol) 7 Plating out transformants on chloroamphenicol plates

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

• Incubation at 37C for 30min
• Heat kill at 80C for 20min

Ligation of Tod genes and pSB1C3 backbone

• For background control, just the pSB1C3 backbone was used
• For positive control, RFP plasmid from transformation efficiency kit provide by iGEM was used
• Quick stick ligase was used and a different protocol from previous day
• Bioline Quick Stick Ligase protocol:
  • • Combine the vector and the insert in the appropriate ratio to make up no more than 100ng of DNA
    • Adjust volume to 14ul with ddH2O
    • Add 1ul of QS ligase
    • Add 5ul of 4x QS Buffer (vortex before use)
  • Mix thoroughly before pipetting
  • Incubate at room temperature for 5min
• The volumes for ligation:

Sample	DNA ul	Vector(pSB1C3) ul	QS ligase ul	Buffer ul	H20 ul
Tod X	0.9	2	1	5	5.1
Tod F	0.8	2	1	5	5.2
Tob B	1	2	1	5	5
Positive control	1	2	1	5	5
Background control	0	2	1	5	6

Transformation of TOD genes ligations with the digested pSB1C3 using the pGEM-T Vector System I-A3600 (changes were made to the protocol)

• Centrifuge the ligations reactions briefly.
• Add 5ul of each ligation reaction to a sterile 1.5ml
• Thaw the competent cells on ice. Mix the cells by gently flicking the tube.
• Carefully transfer 50ul of the competent cells to the ligation reaction tubes.
• Incubate the tubes on ice for 20 minutes
• Heat-shock the cells for 45-50 seconds in a water bath at exactly 42C degrees. Do not Shake. Immediately return the tubes to ice for 2 minutes.
• Add 900ul of room temperature SOC medium to the ligation reaction transformations. incubate for 1 hour at 37C with shaking.

Plating out transformants on chloroamphenicol plates

• Plating out a total of 10 plates, 2 for each sample
• 20ul and 200ul were plated from each sample
• Plates were left over night at 37C.