Paris/July 24
From 2007.igem.org
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(→Purification of PCR Products) |
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* RBS-DapASubtilis = P9 | * RBS-DapASubtilis = P9 | ||
* Lox71-FtsA-FtsZ = P10 | * Lox71-FtsA-FtsZ = P10 | ||
| + | * DGAT1 = P3 | ||
| + | * DGAT2 = P4 | ||
Revision as of 11:14, 31 July 2007
Contents |
Minipreps
BBa_E0422 in pSB1A2 - Clone 1&2 - MP6.1' & MP6.2'
BBa_E0241 in pSB1A2 - Clone 1 - MP7.1'
BBa_J61047 in pSB1A2 - Clone 1&2 - MP9.1' & MP9.2'
Ligation & transformation reactions
| Ligations | |||||
|---|---|---|---|---|---|
| Number | Insert | Insert Volume | Vector | Vector Volume | Comments |
| L1 | D23 (AraC/pBad promoter FI) | D9 (J61002 ready for insertion of a FI) | |||
| L2 | D18 (BB dig. lox66DapAE.coli) | D22 (pSB1A2 Eco, Pst) | |||
| L3 | D19 (BB dig. lox66DapAsubtilis) | D22 (pSB1A2 Eco, Pst) | |||
| L4 | D20 (Lox66-DapAE.coli BI) | D1 (pJ23100 BV) | |||
| L5 | D21 (lox66-DapAsubtilis BI) | D1 (pJ23100 BV) | |||
| L6 | D20 (Lox66-DapAE.coli BI) | D3 (pJ23107 BV) | |||
| L7 | D21 (lox66-DapAsubtilis BI) | D3 (pJ23107 BV) | |||
| L8 | D14 (Cre ORF) | D15 (B0030 BV) | |||
| L9 | D27 (Lox71-ftsZ BI) | D1 (pJ23100 BV) | |||
| L10 | D27 (Lox71-ftsZ BI) | D3 (pJ23107 BV) | |||
- lox66 (annealing O14 & O15) & lox71 (annealing O16 & O17) in D22 (pSB1A2 Eco+Pst)
Annealing of Lox66 and Lox71
Lox66 and Lox71 oligos were designed to form a double-stranded DNA. The extremities bear cohesive overhangs corresponding to digestion by EcoRI and SpeI. See oligos 14 + 15 and 16 + 17 here.
Annealing mix:
- 8 μL of each of the concentrated primers
- 4 μL of salt solution (10 mM NaCl)
- 20 μL of water
Mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally.
More information [http://openwetware.org/wiki/Annealing_complementary_primers here]
Purification of PCR Products
- RBS-DapAColi = P8
- RBS-DapASubtilis = P9
- Lox71-FtsA-FtsZ = P10
- DGAT1 = P3
- DGAT2 = P4
