Matt Gethers/CRI, Thailand/Labwork/Digests/NdeI and BamHI Double Digest

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Contents

NdeI and BamHI Double Digest

Reaction Mix

Reagent Volume
Template 10 μl
NdeI 0.5 μl
BamHI 0.5 μl
Fast Digest Buffer2 μl
H207.0 μl
Total 20 μl

Reaction Conditions

Incubate mixture at 37oC for 30 minutes and inactivate at 65oC for 15 minutes on the heat block.

Run Notes

6.17.08

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.

6.20.08

Followed protocol as written except ~40 minute incubation time to digest HmgR ORF. Inactivation time was 15 minutes as usual.

7.24.08

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.

8.2.08

Followed protocol exactly as written to digest HmgR ORF and pET-11a.Gel Results here.

8.5.08

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.

8.6.08

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.

References

BamHI

NdeI

Double Digest

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