Matt Gethers/CRI, Thailand/Labwork/Gels/Week of 6.15.08

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Contents

6.16.08

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5
5 μl λ DNA Ladder10 μl 1033/1032 PCR Product10 μl 1033/2724 PCR Product10 μl NcoI Digest (of 1033/2724) Product I10 μl NcoI Digest (of 1033/2724) Product I

1%, 90 volts, 45 minutes. I see neither PCR product nor digest product. My positive control failed - that's a problem. I'll talk with Sopapan about the protocol she used to amplify the 1032/1033 fragment and compare with mine to see what exactly I'm failing to do. There's also the possibility that I'm running the gel improperly (insufficient product, improper staining, etc.).

6.17.08

Lane 1 Lane 2 Lane 3
5 μl λ DNA Ladder10 μl 1033/1032 PCR Product10 μl 1033/2724 PCR Product

1%, 100 V, 30 minutes. Both the 1032/1033 and 1033/2724 products worked though there are some funky bands in the 1032/1033 lane - I'm going to chalk it up to non-specific binding aggravated by a long extension time. The product of primary concern, though, seems to be OK.

6.17.08

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6
5 μl λ DNA Ladder10 μl 1033/2724 PCR Product10 μl 2736/2737 PCR Product20 μl NcoI digest of 1033/2724 product20 μl NdeI/BamHI digest of 2736/2737 PCR Product20 μl NdeI/BamHI digest of pET-11a.

1%, 100 V, 30 minute; 20 minute in EtBr. In lane 4, excised the band at 1024 (between the 3rd and 4th bands from the bottom on the marker). In lane 5, excised band at 804 (between the 1st and 2nd band from the bottom on the maker). In lane 6, excised the band at 5637 (between 1st and 2nd band from the top). Used the Quiagen gel clean-up and eluted everything in elution buffer (as opposed to water).

6.18.08

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Lane 9 Lane 10
5 μl λ DNA Ladder5 μl 2736/2737 PCR Product, 50oC annealing temp5 μl 2736/2737 PCR Product, Intermediate annealing temp5 μl 2736/2737 PCR Product, Intermediate annealing temp5 μl 2736/2737 PCR Product, Intermediate annealing temp5 μl 2736/2737 PCR Product, Intermediate annealing temp5 μl 2736/2737 PCR Product, Intermediate annealing temp5 μl 2736/2737 PCR Product, Intermediate annealing temp5 μl 2736/2737 PCR Product, 65oC annealing temp5 μl 1033/2724 PCR Product

1%, 100 V, 30 minutes, stained for 25 minutes. Wow - gel looks good. All annealing temperatures and the positive control worked with all bands at the appropriate locations. I excised the bands and did a gel clean up just in case the PCR fails again just as cryptically as it started working.

|5 μl λ DNA Ladder||10 μl 1033/1032 PCR Product||10 μl 1033/2724 PCR Product||10 μl NcoI Digest (of 1033/2724) Product I||10 μl NcoI Digest (of 1033/2724) Product I |}

1%, 90 volts, 45 minutes. I see neither PCR product nor digest product. My positive control failed - that's a problem. I'll talk with Sopapan about the protocol she used to amplify the 1032/1033 fragment and compare with mine to see what exactly I'm failing to do. There's also the possibility that I'm running the gel improperly (insufficient product, improper staining, etc.).

6.17.08

Lane 1 Lane 2 Lane 3
5 μl λ DNA Ladder10 μl 1033/1032 PCR Product10 μl 1033/2724 PCR Product

1%, 100 V, 30 minutes. Both the 1032/1033 and 1033/2724 products worked though there are some funky bands in the 1032/1033 lane - I'm going to chalk it up to non-specific binding aggravated by a long extension time. The product of primary concern, though, seems to be OK.

6.20.08

Lane 1 Lane 2
5 μl λ DNA Ladder20 μl NdeI/BamHI digest of HmgR ORF.

1%, 100 V, 30 minutes; 15 minutes staining. Expect band at ~800 bp. Found band at correct length and excised. Did gel purification and eluted in elution buffer.

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