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Team:Hong Kong CUHK/july
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Revision as of 06:00, 22 October 2013
July
July 2
1. Restriction digestion of T7RBS plasmid with E and P in buffer 2. Prepared by BL21 and DH5
2. Run gel in the following order:
Ladder (100-6000bp), new T7-RBS complete, new T7-RBS cut, old T7-RBS complete, old T7-RBS cut, psB1K3+RFP complete.
Seems like it fails as well. We may need to redo transformation protocol for T7-RBS.
> Gel photo 2
July 3
1. Transformation of T7-RBS into DH5
2. Transformation of pSB1C3-RFP into DH5 and BL21 to test for competency.
July 4
1. pSB1C3-RFP in DH5 gave false clone, and there were no colony in BL21 plate, so the cells were bad. We need to redo competent cells again.
2. T7-RBS in DH5 shows colonies with a lot of satellites. Large colonies were picked out of each plate and shaked with C antibiotic.
3. DGS students came to lab for lab visit. They saw demonstration of picking colonies.
July 5
1. Finished preparation of T7-RBS.
2. CCMB80 prepared.
3. Ran gel with restriction digestion with EcoRI and PstI confirmed presence of T7-RBS.
> Gel photo 3
July 8
Streak plates with DH5 and BL21.
July 9
Pick colonies from DH5 and BL21 plates. 2 flasks of LB(each 275ml) is being autoclaved for tomorrow use.
July 10
Competent cells (BL21 + DH5 ) made.
Transformation with pSB1C3+RFP.
July 11
Picked colonies and shaked overnight from pSB1c3_RFP in old DH5 . There was no result from the old BL21 and the new completent cells will try again on monday.
July 12
Miniprep and ran gel with pSB1C3_RFP. Correct bandings were observed with an unknown band at 6000 or higher.
>Gel photo 4
July 15
Transformation on 23100 nto DH5 .
July 16
PCR with primers of Lgt.
Ran gel, a band of 100bp obtained which is not the desired band.
Slight band smaller than 100bp is from primers, bright band of around 100bp is from self annealing of the reverse single strand of around 1000bp probably.
>Gel photo 5
July 17
Human practice: Lab workshop and talk.
>Gel photo 6
>Gel photo 7
July 19
Restriction digestion and run gel, no bands showed up. It was later found that C18 from plate 1 is used, and the J23100 is in plate 5.
>Gel photo 8
July 22
Transformation with J23100.
Prepare for lab workshop.
July 23
The plate was full of bacteria, probably because antibiotic is added too early when the agar is still too hot. Rehearsal for lab workshop.
July 24
Prepare for workshop.
Picking colonies, only 5 colonies are found in old plate.
July 25
Human practice: workshop and talk.
Miniprep.
July 26
Run gel, restriction digestion. Get no result.
Move all stuff in BME lab back. Move PCR machine to lab.
July 29
Transformation:
A: J23100 from plate 5, J23100 provided by Isaac, M2 from SJTU, M3 from SJTU;
C: RFP (test competency)
Streak plate for making competent cells.
July 30
Prepare buffer for making competent cells.
Pick colonies.
Transformation:
A: M2, M3, 23100, QsrR
C: RFP (test)
K: Laccase, Dioxygenase
July 31
Transformation: M2,M3, 23100, QsrR, RFPx2(Test) (failed)
Pick colonies: Laccase, Dioxygenase
Miniprep of M2x2+23100+QsrR and gel electrophoresis (no result)
Make competent cells.
