Jessica Karen Wong/Notebook/2007-6-21
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To Do
- Get Jason to log in to point mutate I2054
- Make plan for next week
- Digest backbones, J04650, B0032, R0040
- PCR T9002, E0240
Digest
- See protocol
- Digested plasmids 1AT3, 1AK3, and 1AC3 with EcoR1 and Pst
- Used 3ul of AT3 and AC3 plasmid and 4ul AK3 to insure 1 ug of DNA
- Used EcoR1 buffer
- Digested B0032 and R0040 with EcoR1 and Spe
- Used 2ul of B0032 DNA 3ul of R0040 DNA
- Used EcoR1 buffer
- Digested J04650 with Xba and Pst
- Used 5ul of prepped DNA
- Used buffer 3 as suggested by NEB for the XbaI/PstI digests
- Incubated at 37C overnight
PCR
- Did a 100ul preparatory PCR on T9002 and E0240
- Based on Endy:Colony PCR
- 95.25 ul PCR Mix
- 25ul Thermo Polymerase Buffer
- 25ul dNTP's (10x)
- 3.75ul Rev Primer (40uM)
- 2.5ul Taq Polymerase
- 183 Water
- 1 ul DNA (3x dilution)
- 3.75 ul forward primer (because T9002 and E0240 have different forward primers but the same reverse primer)
- 95.25 ul PCR Mix
- Dilute primers to 40 uM
- Added 730ul water to E0240F
- Added 821ul to T9002F
- Added 925ul to E0240R
- Did a 3x dilution of DNA samples
Sequencing
- Nishant prepped and sent I0255 to be sequenced