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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.
Munima
Objective: Subculture cells from transformation to determine if it was successful
Inoculated three colonies from transformation done yesterday (by Roxanne) of DH5-alpha + pE43 into LB + Tet (100 ug/mL). Tubes were put into the shaker incubator at 37 C overnight.
October 11, 2008
-Ran gel of the restriction digested xylE amplicon and the pE43 plasmid prep.
-PCR of the ohbA, ohbB, ohbC, ohbR genes from pE43 with an annealing temperature of 51.0 C.
-Ran a gel of the ohb gene amplicons
-Restriction digested the ohb genes with XbaI and PstI overnight at 37.0C
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.
