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Team:University of Lethbridge/Notebook/Project4October
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October 6, 2008
Jaden
Objective: Grow up cells containing the xylE gene from the glycerol stocks and plates (Selina had transformed with DH5a) in LB + Amp liquid media. The gene was sent to us in pVL1392 (modified pUC8 for viral expression, with Amp resistance).
Subcultured four tubes: Two from glycerol stocks and two from colonies on a plate.
October 7, 2008
Christa, Munima
Objective: Plasmid prep the xylE gene that Jaden subcultured yesterday.
Christa: Plasmid prepped one subculture tube from 'glycerol stock' and 'plate' using the Eppendorf FastPlasmid Mini Prep Kit. Stored 50 uL aliquots in the -20 C iGEM freezer box (iGEM Plasmids) labeled "xylE from glycerol stock" and "xylE from plate", and have a big blue "P" or "G" on the top, respectively.
Munima: Plasmid prepped one subculture tube from 'glycerol stock' and 'plate' using the QiaPrep Spin MiniPrep Kit. Plasmids are stored in 50 uL aliquots in the -20 C iGEM freezer in the iGEM Plasmids box. They are labeled "xylE plasmid; plate 2; Oct. 7/08" and "xylE plasmid; glycerol 2; Oct. 7/08".
October 8, 2008
Christa, Roxanne
Ran gel to assess if gel extractions and plasmid preps worked. See gel for results. Plasmid preps of xylE yielded no results with the Eppendorf Kit, so use Qiagen kit from now on.
October 10, 2008
Roxanne
Objective: Set up PCR for xylE gene with Phusion enzyme.
Master Mix for 5 reactions with Phusion:
- 10x Buffer: 25 uL - 10 mM dNTPs: 5 uL - 50 mM Mg2+: 7.5 uL - 10 uM RF: 5 uL - 10 uM RR: 5 uL - Phusion polymerase: 1 uL - H20: 196.5 uL - Total volume is 245 uL. Therefore, 49.0 uL in each reaction tube. - template: 1 uL into each reaction tube to equal 50 uL volume
Cycle conditions:
1. Denaturation: 94 C for 1 min 2. a. Denaturation: 94 C for 30 seconds b. Annealing: 52 C for 30 seconds c. Extension: 70 C for 30 seconds Repeat Step 2 for 30 cycles 3. Final extension: 72 C for 10 minutes, then hold at 4 C.
October 11, 2008
Roxanne
-Ran a gel of the PCR products to determine the result of the PCR reaction.
-Set up a Restriction Digest of the xylE gene and pSB1A2 for GFP sub, using XbaI and PstI. Incubate overnight at 37.0C.
