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Team:TU Munich/Notebook/Labjournal/Week 1
From 2012.igem.org
Contents |
Wednesday, June 13th
Exchange of the Multiple Cloning Site of pYES2
Investigator: Saskia, Daniela
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
Hybridisation of the primers O1 with O2 and O3 with O4
Primer preparation:
- centrifugation
- dilution in the denoted quantity in bidest. water (concentration = 100 pmol/µl)
- centrifugation
Hybridisation:
| volume | reagent |
| 34 µl | ddH2O |
| 5 µl | PNK-buffer |
| 2.5 µl | Primer O1 |
| 2.5 µl | Primer O2 |
| 1 µl | PNK (10mM) |
| volume | reagent |
| 34 µl | ddH2O |
| 5 µl | PNK-buffer |
| 2.5 µl | Primer O3 |
| 2.5 µl | Primer O4 |
| 1 µl | PNK (10mM) |
- 30 min 37 °C
- 10 min 80 °C
- put the Thermo Block with the mixture in a styrofoam box and let it cool down over night
Thursday, June 14th
Exchange of the Multiple Cloning Site of pYES2
Investigator: Saskia, Daniela
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
Digestion of pYES2 with HindIII and XbaI
| volume | reagent |
| 12 µl | Miniprep (pYES2 SH 1.7.3 with a concentration of 87.8 ng/µl) |
| 5 µl | NEB2 |
| 5 µl | 10x BSA |
| 2 µl | XbaI (10 U/µl) |
| 2 µl | HinIII (10U/µl) |
| 24 µl | ddH2O |
Incubation: 37 °C, 1.75 h
DNA preparative gel electrophoresis
- gel: 1% with LMP-agarose
- band 1: 10 µl DNA ladder (1kb)
- band 2: 50 µl probe + 5 µl loading dye
- 70 V, 90 min
Gelextration
- cut the bands (5.7-5.8 kb) and split it in two eppis
- m1=165.3 mg
- m2=211.1 mg
- QIAquick Gel Extractrion Kit
- eppi1: 495.9 µl QG-buffer
- eppi2: 633.3 µl QG-buffer
- step 6 was left out
- step 9: 30µl buffer, 4 min incubation
- the product was named P5
Transformation of plasmids from Prof. Schwab in E.coli XL-1 Blue
Investigator: Lara, Andrea
Aim of the experiment: Preparation of the plasmids for transformation
Overnight cultures of cells with limonenesynthase-plasmid from Prof. Schwab
- resuspend 50 µl / 200 µl of competent cells with 50 ml LB medium
- add 0,1 ml Ampicillin (100 µg/ml) and 0,28 ml Chloramphenicol (170 µ/ml) for strain 108
- add 0,07 ml Kanamycin (50 µg/ml) and 0,28 ml Chloramphenicol (170 µ/ml) for strain 106
- incubate at 37 °C
Friday, June 15th
Exchange of the Multiple Cloning Site of pYES2
Investigator: Saskia, Daniela
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
Analytical DNA gel electrophoresis
- gel: 1.2 %
- band 1: 10 µl DNA ladder (1kb)
- band 2: 3 µl pYES2 digested + 7 µl TAE-buffer + 1 µl loading dye
- band 3: 3 µl O5 + 7 µl TAE-buffer + 1 µl loading dye
- band 4: 3 µl O6 + 7 µl TAE-buffer + 1 µl loading dye
- band 5: 10 µl DNA ladder (100 bp)
Ligation of Plasmid P5 (pYES2 digested) with the hybridized primers O5 and O6
| volume | reagent |
| 4 µl | pYES2 digested (P5) |
| 1 µl | O5 |
| 1 µl | O6 |
| 2 µl | T4-ligase buffer (10x) |
| 0,5 µl | T4 DNA-ligase |
| 11.5 µl | ddH2O |
Negative control
| volume | reagent |
| 4 µl | pYES2 digested (P5) |
| 2 µl | T4-ligase buffer (10x) |
| 0,5 µl | T4 DNA-ligase |
| 13.5 µl | ddH2O |
- water bath 16 °C
- after 3 h a probe for the transformation was taken
- the rest was ligated over the weekend
Transformation of E. coli with ligated products (P6)
- competent cells: SHXL1 Blue (by Simon)
- Transformation with ligation product (P6) and negative control
results:
- P6 (100 µl): 1 clone
- P6 (concentrated): 30 clones
- negative control (100 µl): 0 clones
- negative control (concentrated): 6 clones
Transformation of plasmids from Prof. Schwab into E.coli XL-1 Blue
Investigator: Andrea
Aim of the experiment: Preparation of the plasmids for transformation
Determination of the concentration with Nano Drop
| Sample | concentration [ng/µl] |
| P3 | 1353 |
| P4 | no result |
- the strain 106 culture was not grown satisfying and were incubated for 2 more days
Miniprep of pGex-4T-1 of strain 108 from Prof. Schwab
- see QIAprep Spin Miniprep Kit
- stored as P3 (-20 °C)
Sunday, June 17th
Exchange of the Multiple Cloning Site of pYES2
Investigator: Saskia, Daniela
Aim of the experiment: Exchange of the Multiple Cloning Site of pYES2
Picking clones for Miniprep
- 10 clones of transformed E.coli with P6 were picked
- medium: 5ml LB with Amp
