Koeris/Notebook/2007-1-18

Transformations

Continued from 1/17. A construct didn't give any colonies -> try using more plasmid DNA, i.e. not 1ul but 2.5ul and 5ul in two aliquots.

Also plate ON RT aliquot, maybe there is one or two transformants, though historically there is a lower chance of that happening.

P. aeruginosa mer operon cloning

Continued from 1/15.

Approach

I designed two primers: one new set of mer operon primers, that are significantly longer than before (merSO 2F&R 44bp, 36bp), as well as a set of check primers to see if the DNA preparation is usable at all, or if it is degraded.

I will use the Invitrogen Taq SuperMix for the PCR.

PCR set-up

• 50 ul total reaction volume
• 1ul template DNA @ 300ng/ul
• 200pmol primer mix or 400pmol
• 47ul Taq SuperMix

Pipetting scheme

Labels in the respective field codes

Primer	Tube Label
merSO 2F&R 200pmol	A1
merSO 2F&R 400pmol	A2
recF 1F&R 200pmol	A3
recF 1F&R 400pmol	A4

Thermal profile

1. 92 deg C - 5min
2. 92 deg C - 30s
3. 50 deg C - 45s
4. 72 deg C - 120s
5. Cycle back to 2. 29X
6. 72 deg C - 10min
7. 4 deg C - indefinite

Gel Run

IMPORTANT: Load genomic DNA on the gel as well to test -> at least 1ug - 2ug

• 14-comb gel @ 50ml
• Loading volume: 20ul of PCR Rx = 40% product
• 1% agarose TAE gel, ran @ 100V for 60min

Lanes

Description

Starting from the ladder

1. Ladder (1ug load total)
2. 1.5ug genomic DNA
3. merSO 2F&R 200pmol
4. merSO 2F&R 400pmol
5. recF 1F*R 200pmol
6. recF 1F&R 400pmol

Gel run shows that the genomic DNA isolated from P. aeruginosa is a valid template for PCR. On the other hand the mer operon cannot be amplified that easily. Check back the genotype of P. aeruginosa with Guillaume.

Also check if the recF primers are able to bind somewhere in K-12 E. coli -> cross-contamination.

Solution

The P. aeruginosa strain we were using is not the correct one. We need PA14, instead we have PAO-1...