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Team:Brown/Notebook/August13
From 2010.igem.org
Friday, August 13, 2010
Gel extractions from yesterday's gel
Gel extracted the following using Qiagen kit:
- CI - 17.3ng/ul
- Mnt - 15ng/ul
- AraC - 14.2ng/ul
- RFC25_AraC (little tube) - 9.5ng/ul
Gel of AraC-RFC25 re-PCR
17ul DNA, 3ul loading buffer
Gel product was extracted in 30ul EB. Final concentration: 14.4ng/ul
Ligations of CI PCR, Mnt PCR, AraC PCR into pGEM
1:3 vector to insert ratio. 15ul total volume:
- 7.5ul ligase buffer (2x)
- 1ul T4 DNA ligase
- 0.5ul vector (pGEM-T-easy)
- 5.5ul DNA
- 0.5ul dH2O?? (was not clear from paper-based notebook)
Control ligation
15ul total volume:
- 7.5ul ligase buffer (2x)
- 1ul T4 DNA ligase
- 0.5ul vector (pGEM-T-easy)
- 1.5ul DNA
- 4.5ul dH2O
Digest of Lovtwo miniprep w/ EcoRI and BglII
50ul total volume:
- 5ul NEB buffer 2
- 2ul DNA
- 0.5ul 100x BSA
- 1ul EcoRI OR BglII
- 41.5ul dH2O
Gel of products
- Ladder - 14ul
- Lovtwo-EcoRI - 13ul
- Lovtwo-BglII - 12ul
- Lovtwo-miniprep - 9ul
PCR of Lovtwo wt into Lovtap2 (Lovtap2 has Bgl sites?)
50ul total volume:
- 2ul fwd primer
- 2ul rev primer
- 1ul template (lovTwo wt miniprep at 192.5ng/ul)
- 20ul dH2O
- 25ul master mix
