Matt Gethers/CRI, Thailand/Labwork/Gels/Week of 6.22.08

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6.23.08

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
5 μl λ DNA Ladder 10 μl 2736/2737 colony PCR Product 1 10 μl 2736/2737 colony PCR Product 2 10 μl 2736/2737 colony PCR Product 3 10 μl 2736/2737 colony PCR Product 4 10 μl 2736/2737 colony PCR Product 5 10 μl 2736/2737 colony PCR Product 6 10 μl 2736/2737 Positive Control (genomic DNA template)

100 V, 30 minutes, 1% gel, 15 minutes staining in EtBr. No product in the colony PCR product lanes (1-6); the positive control lane has a product in it, but it was significantly larger than it should be (near the 21 Kb band). The PCR protocol I used was the same as the other I used to amplify the HmgR ORF, except that the extension time was reduced to 2 minutes. I used the PCR supermix as well, however. A puzzle...

6.24.08

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
5 μl λ DNA Ladder 10 μl 2736/2737 colony PCR Product 1 (Pfu) 10 μl 2736/2737 colony PCR Product 2 (Pfu) 10 μl 2736/2737 colony PCR Product 3 (Pfu) 10 μl 2736/2737 colony PCR Product 4 (Pfu) 10 μl 2736/2737 colony PCR Product 5 (Pfu) 10 μl 2736/2737 Positive Control (genomic DNA template, Pfu) 10 μl 2736/2737 Positive Control (genomic DNA template, supermix)

100 Volts, 30 minutes, 1% gel, stained for 20 minutes in EtBr. Surprise! Colony 5 product at ~800 bp. This didn't show up when I used the supermix. Also, the positive control lanes differ - the Pfu gives the correct product while the supermix still gives that very large band (between 5 and 20 Kb). Interesting. I don't know that I can trust the supermix.

6.25.08

Gel 1

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
5 μl λ DNA Ladder 10 μl 2736/2737 colony PCR Product 1 (Supermix) 10 μl 2736/2737 colony PCR Product 2 (Supermix) 10 μl 2736/2737 colony PCR Product 3 (Supermix) 10 μl 2736/2737 colony PCR Product 4 (Supermix) 10 μl 2736/2737 colony PCR Product 5 (Supermix) 10 μl 2736/2737 colony PCR Product 6 (Supermix) 10 μl 2736/2737 Positive Control (genomic DNA template, supermix)

100 Volts, 30 minutes, 1% gel, stained for 20 minutes in EtBr. Nothing showed up even with the new aliquot of supermix. Boo, supermix.

Gel 2

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
5 μl λ DNA Ladder 5 μl M13_for/BT2724 colony PCR Product 1 (Supermix) 5 μl M13_for/BT2724 colony PCR Product 1 (Supermix) 5 μl M13_for/BT2724 colony PCR Product 1 (Supermix) 5 μl M13_for/BT2724 colony PCR Product 1 (Supermix) 5 μl M13_for/BT2724 colony PCR Product 1 (Supermix) 5 μl M13_for/BT2724 colony PCR Product 1 (Supermix) 5 μl 2736/2737 Positive Control (genomic DNA template, supermix)

100 Volts, 30 minutes, 1% gel, stained for 20 minutes in EtBr. Nothing showed up even with the new aliquot of supermix. I kind of expected this result seeing as how the previous supermix PCR failed, but I had started this PCR before I had the results of the previous one.

6.26.08

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Lane 9 Lane 10 Lane 11 Lane 12 Lane 13 Lane 14
5 μl λ DNA Ladder 5 μl M13_for/BT2724 colony PCR Product 1 (Taq) 5 μl M13_for/BT2724 colony PCR Product 2 (Taq) 5 μl M13_for/BT2724 colony PCR Product 3 (Taq) 5 μl M13_for/BT2724 colony PCR Product 4 (Taq) 5 μl M13_for/BT2724 colony PCR Product 5 (Taq) 5 μl M13_for/BT2724 colony PCR Product 6 (Taq) Blank 5 μl 2736/2737 colony PCR Product 1 (Taq) 5 μl 2736/2737 colony PCR Product 2 (Taq) 5 μl 2736/2737 colony PCR Product 3 (Taq) 5 μl 2736/2737 colony PCR Product 4 (Taq) 5 μl 2736/2737 colony PCR Product 5 (Taq) 5 μl 2736/2737 Positive Control (genomic DNA template, Taq)

1% gel, 100 V, 30 minutes, 20 minute in EtBr. It looks like the HmgA upstream fragment (1 by PiSo's numbering) ligation into pUC18 worked. 5 of the 6 colonies I screened (lanes 2-3, 5-7) showed up as having a product at the correct length. There were some other faint bands, but the very bright ones were at the expected length. The pET-11a/HmgR colonies, however, still didn't yield anything (lanes 9-13) while the positive control worked faintly.

6.27.08

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
5 μl λ DNA Ladder 5 μl 2736/2737 colony PCR Product (#5) (Taq) 5 μl 2736/2737 Positive Control (genomic DNA template, Taq) 5 μl pET-11a/HmgR ORF miniprep (1 μl product, 4 μl H20) 5 μl pUC18+HmgR upstream mix (1 μl each, 3 μl H20) 5 μl pUC18/HmgR upstream fragment ligation product (2 μl product, ,3 μl H20) 5 μl pET-11a+HmgR ORF(1 μl each, 3 μl H20) 5 μl pET-11a/HmgR ORF ligation product (2 μl product, 3 μl H20)

100 Volts, 1% gel, 10 minutes, 30 minutes staining. Neither the colony PCR product nor the positive control showed up on the gel, so still I have neither a confirmation nor a refutation of my one positive result for colony 5. The prep of pET-11a/HmgR ORF has about the same amount of DNA as the 1.5 Kb band - ~15 ng/0.5 μg of ladder run in a lane. I need to figure out how much ladder is in the 5 μl PiKoy generally loads. The pUC18/HmgR upstream fragment ligation product looks very different from the pUC18/HmgR mix; I hope that's indicative of a good ligation. The pET-11a/HmgR ORF ligation product is very faint if it's there at all and I don't see the bands that are present in the pET-11a/HmgR ORF mix. I'm not sure what to make of that.