Matt Gethers/CRI, Thailand/Labwork/Digests/NdeI and BamHI Double Digest
NdeI and BamHI Double Digest
|Fast Digest Buffer||2 μl|
Incubate mixture at 37oC for 30 minutes and inactivate at 65oC for 15 minutes on the heat block.
Followed protocol as written to digest HmgR ORF and vector pET-11a. I first digested HmgR, inactivated, then placed in the 4oC for ~1 hour while I digested pET-11a.
Followed protocol as written except ~40 minute incubation time to digest HmgR ORF. Inactivation time was 15 minutes as usual.
45 minute incubation time to digest the HotStar HmgR ORF. Inactivation time was 10 minutes. I did a PCR clean up rather than running a gel to get rid of the small fragments. Eluted in 10 μl EB.
Redigesting pET-11a the the HmgR ORF with some controls. See reaction mixes. Incubated for 55 minutes at 37 degrees, then ran immediately on a 1% agarose gel (no 65 degree inactivation) and excised bands. Did a gel clean-up and eluted pET-11a (NdeI/BamHI) and HmgR ORF (NdeI/BamHI) in 50 μl elution buffer. Gel results here.
Redigesting the HmgR ORF from 8.2.08, but this time I neither ran out on a gel nor used a PCR clean up. I think both processes cause me to lose too much insert and are resulting in an inefficient ligation. I used 10 μl template, 2 μl buffer, 0.5 μl of each enzyme, and 7 μl water. I digested for 45 minutes at 37 degrees and I did inactivate at 65 degrees for 15 minutes before using in the ligation.