SEED/2008/Day 4: Difference between revisions
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== Morning == | == Morning == | ||
* Set up 50ul PCRs with 2 different forward RBS primers | |||
** 5ul 10x NovaTaq Buffer with MgCl<sub>2</sub> | |||
** 1ul dNTP mix (10mM each dNTP) | |||
** 1ul reverse primer (VR) | |||
** 1ul RBS specific forward primer | |||
** 0.25ul NovaTaq DNA polymerase | |||
** 0.5ul DNA template of E0050 (10ng) | |||
** rest PCR grade water | |||
* Cycle 30 times | |||
** 30s@94C | |||
** 30s@55C | |||
** 1m@72C | |||
* Pour gel | * Pour gel | ||
== Afternoon == | == Afternoon == | ||
* Cut out bands and | * Run plasmid digest and PCRs on gel | ||
* Cut out bands and save in freezer | |||
== Instructor Preparation == | == Instructor Preparation == | ||
* Oligos | |||
** VR: attaccgcctttgagtgagc | |||
** B0030.E0040-F: gaattctctagagattaaagaggagaaatactagatgcgtaaaggagaagaacttttc | |||
** B0031.E0040-F: gaattctctagagtcacacaggaaacctactagatgcgtaaaggagaagaacttttc | |||
** B0032.E0040-F: gaattctctagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttc | |||
** B0033.E0040-F: gaattctctagagtcacacaggactactagatgcgtaaaggagaagaacttttc | |||
** B0034.E0040-F: gaattctctagagaaagaggagaaatactagatgcgtaaaggagaagaacttttc | |||
** B0035.E0040-F: gaattctctagagattaaagaggagaatactagatgcgtaaaggagaagaacttttc | |||
* Pairs of RBS primers to assign to groups (selected based on estimated strength): | |||
** B0030 & B0032 | |||
** B0031 & B0035 | |||
** B0033 & B0034 | |||
** B0032 & B0033 | |||
** B0031 & B0034 | |||
** B0030 & B0035 | |||
Revision as of 16:22, 7 March 2008
Morning
- Set up 50ul PCRs with 2 different forward RBS primers
- 5ul 10x NovaTaq Buffer with MgCl2
- 1ul dNTP mix (10mM each dNTP)
- 1ul reverse primer (VR)
- 1ul RBS specific forward primer
- 0.25ul NovaTaq DNA polymerase
- 0.5ul DNA template of E0050 (10ng)
- rest PCR grade water
- Cycle 30 times
- 30s@94C
- 30s@55C
- 1m@72C
- Pour gel
Afternoon
- Run plasmid digest and PCRs on gel
- Cut out bands and save in freezer
Instructor Preparation
- Oligos
- VR: attaccgcctttgagtgagc
- B0030.E0040-F: gaattctctagagattaaagaggagaaatactagatgcgtaaaggagaagaacttttc
- B0031.E0040-F: gaattctctagagtcacacaggaaacctactagatgcgtaaaggagaagaacttttc
- B0032.E0040-F: gaattctctagagtcacacaggaaagtactagatgcgtaaaggagaagaacttttc
- B0033.E0040-F: gaattctctagagtcacacaggactactagatgcgtaaaggagaagaacttttc
- B0034.E0040-F: gaattctctagagaaagaggagaaatactagatgcgtaaaggagaagaacttttc
- B0035.E0040-F: gaattctctagagattaaagaggagaatactagatgcgtaaaggagaagaacttttc
- Pairs of RBS primers to assign to groups (selected based on estimated strength):
- B0030 & B0032
- B0031 & B0035
- B0033 & B0034
- B0032 & B0033
- B0031 & B0034
- B0030 & B0035
