Koeris/Notebook/2007-1-15

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
• 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

1. Primers (F&R): 20 ul
2. Taq polymerase standard reaction buffer (10X): 50 ul
3. dNTP mix (40mM): 10 ul, i.e. final conc. 4mM/Rx
4. Taq DNA polymerase (5U/ul): 5 ul, i.e. final conc. 0.5U/Rx
5. ddH₂O: 320 ul

Master mix (4 pmol primers) 10X

1. Primers (F&R): 40 ul
2. Taq polymerase standard reaction buffer (10X): 50 ul
3. dNTP mix (40mM): 10 ul, i.e. final conc. 4mM/Rx
4. Taq DNA polymerase (5U/ul): 5 ul, i.e. final conc. 0.5U/Rx
5. 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

1. 92 deg C - 5min
2. 92 deg C - 30s
3. 50 deg C - 45s
4. 72 deg C - 120s
5. 4 deg C - indefinite

Gel image

1% agarose TAE gel, ran @ 100V for 60min