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Team:TU Munich/Notebook/Labjournal
From 2012.igem.org
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Revision as of 16:40, 23 June 2012
Contents |
Friday, 22th June
Transformation of E.coli XL1-Blue with pKS2µHyg-PAL-4Cl-CHS
Investigator: Ingmar, Volker
Aim of the experiment: Plasmid amplification
Operation Sequence:
- melting of 100 µl Ca-competent E.coli XL1-Blue cells
- addition of 1 µl of the Plasmid pKS2µHyg-PAL-4Cl-CHS
- incubation for 30 min on ice
- heat shock for 5 min at 37 °C
- transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
- plate 100 µl on an Amp-LB-plate
- sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate
Saturday, 23th June
Quick Change mutagenis to remove NgoMIV from pYES2
Investigator: Ingmar, Volker
Aim of the experiment: Generation of an RFC 25 compatible version of the pYes2 Vector.
PCR
Reaction batch
| volume | reagent |
| 2.5 µl | 10x Pfu Ultra II buffer |
| 4 µl | Plasmid P7 pYes2_RFC25 MCS template |
| 0.5 µl | 1:10 dilution of O38 (10 pmol/µL) |
| 0.5 µl | 1:10 dilution of O39 ((10 pmol/µL) |
| 17 µl | ddH2O |
| 0.5 µl | dNTP mix |
| 0.5 µl | Pfu Turbo DNA polymerase (2.5 U / µl) |
PCR cycling parameters
| Segment | Cycles | Temperature | Time |
| 1 | 1 | 95 °C | 30 sec |
| 2 | 15 | 95°C | 30 sec |
| 55°C | 1 min | ||
| 68°C | 6 min |
===Transformation with E.coli Xl1-Blue===
