User:Mbennie/Notebook/Lab Notebook/Notebook/2008/01/23: Difference between revisions
From OpenWetWare
| Line 19: | Line 19: | ||
** Template for assembly: .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn) | ** Template for assembly: .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn) | ||
** Protocol: 30mins@roomtemp,10mins@65C | ** Protocol: 30mins@roomtemp,10mins@65C | ||
* Transformation | |||
** 50ul of TOP10 cells on ice with 2ul of DNA for 30mins | |||
** 45secs heat shock at 42C using water bath | |||
** Added 300ul of SOC | |||
** Incubated for 1hr at 37C | |||
** Plated on Amp/Cl and grew up at 37C overnight | |||
Revision as of 14:10, 23 January 2008
Cellular Adhesion
- Gel
- Ran 1% gel for 25 minutes at 95V with samples (5ul sample with 2ul of loading dye)
- Not a great gel, but the smudges around 100 bp look promising
- Extra band in GCN4 sample
- PCR Purification
- Used MinElute columns to purify the five tags and one zipper from 1.22 PCR
- Eluted in 10ul of water
- Digest
- Ran on PCR purification above
- Template for tags: 3ul DNA (each), 2ul NEB2, .5ul EcoR1, .5ul Pst1, rest water (20ul rxn)
- Thermocycler protocol: 1hr@37C, 20mins@80C
- Ligation
- Ran on digest samples above
- Template for assembly: .2ul 1AC3 vector, 1ul ligation buffer, .2ul ligase, 1ul digest product, rest water(10ul rxn)
- Protocol: 30mins@roomtemp,10mins@65C
- Transformation
- 50ul of TOP10 cells on ice with 2ul of DNA for 30mins
- 45secs heat shock at 42C using water bath
- Added 300ul of SOC
- Incubated for 1hr at 37C
- Plated on Amp/Cl and grew up at 37C overnight
