Koeris/Notebook/2007-1-15: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 65: | Line 65: | ||
#50 deg C - 45s | #50 deg C - 45s | ||
#72 deg C - 120s | #72 deg C - 120s | ||
#Cycle back to | #Cycle back to 2 | ||
#72 deg C - 10min | #72 deg C - 10min | ||
#4 deg C - indefinite | #4 deg C - indefinite |
Revision as of 13:40, 15 January 2007
Pseudomonas aeruginosa mer operon cloning 3
Preparation
What can I do in order to make sure the cloning works this time?
- Check concentrations of the chromatin DNA: 351 ng/ul
Remember its not pure DNA but rather cracked open cells, still yield is appropriate.
- Do a dilution series over two orders of magnitude of the template DNA: 3ng/ul - 300ng/ul
- All dilutions are to be done in at LEAST 100ul volume ddH2O
- Dilute stock 1:1.2: yields 300ng/ul
- Dilute twice 1:10: yields 30ng/ul and 3ng/ul
PCR set-up of the operon
50 ul total reaction volume
Master mix (2 pmol primers) 10X
- Primers (F&R): 20 ul
- Taq polymerase standard reaction buffer (10X): 50 ul
- dNTP mix (40mM): 10 ul, i.e. final conc. 4mM/Rx
- Taq DNA polymerase (5U/ul): 5 ul, i.e. final conc. 0.5U/Rx
- ddH2O: 320 ul
Master mix (4 pmol primers) 10X
- Primers (F&R): 40 ul
- Taq polymerase standard reaction buffer (10X): 50 ul
- dNTP mix (40mM): 10 ul, i.e. final conc. 4mM/Rx
- Taq DNA polymerase (5U/ul): 5 ul, i.e. final conc. 0.5U/Rx
- ddH2O: 300 ul
Pipetting scheme
Labels in the respective field codes
Template DNA concentration | Primerconcentration [2 pmol] | Primerconcentration [4 pmol] |
---|---|---|
3 ng | A1 | B2 |
6 ng | A2 | B2 |
9 ng | A3 | B3 |
30 ng | A4 | B4 |
60 ng | A5 | B5 |
90 ng | A6 | B6 |
150 ng | A7 | B7 |
300 ng | A8 | B8 |
Thermal profile
- 92 deg C - 5min
- 92 deg C - 30s
- 50 deg C - 45s
- 72 deg C - 120s
- Cycle back to 2
- 72 deg C - 10min
- 4 deg C - indefinite
Gel image
- Two 14-comb gels @ 50ml
- Loading volume: 20ul of PCR Rx = 40% product
- 1% agarose TAE gel, ran @ 100V for 60min