Matt Gethers/CRI, Thailand/Labwork/Digests/SphI-HindIII

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SphI and HindIII Double Digest

Reaction Mix

Reagent Volume
Template 10 μl
SphI 0.5 μl
HindIII 0.5 μl
Fast Digest Buffer 2 μl
H20 7.0 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

7.4.08

I ran a digest on pKn002.P1 using only 5 μl of prep and 12 μl of water. Other reagents were added as written. Incubated for 1 hour at 37oC, 20 minutes at room temp, inactivated at 65oC for 15 minutes. This digest will result in one 10 bp fragment and one ~4.2 kb fragment. I used a gel excision to purify the product.

7.10.08

I ran another digest on pKn002.P1 using only 5 μl of prep and 12 μl of water. Other reagents were added as written. Incubated for 1 hour at 37oC, 20 minutes at room temp, inactivated at 65oC for 15 minutes. This digest will result in one 10 bp fragment and one ~4.2 kb fragment. I used a PCR clean up rather than a gel to purify this time around. Eluted in 10 μl elution buffer.

7.21.08

I ran another digest on pKn002.P1. P'So suggested my cutting inefficiency may be due to competition between the enzymes for space on the DNA (cut sites only 10 bp apart) and that I might try digesting serially. I set up five 20 μl reactions with 2 μl vector, 2 μl buffer, and water up to 19 μl in each: No enzyme (negative control), SphI only, HindIII only, SphI/HindIII simultaneously added, and SphI/HindIII added serially and in that order. For all reactions, I added the appropriate enzymes and incubated for 1 hour at 37oC. For the last reaction (serial digest), I digested with SphI for one hour, then added HindIII and incubated for one more hour. After all digestion was complete, I heated at 65oC for 15 minutes. I'll run out on a gel tomorrow to compare the digestions.

References

Fast Digest HindIII

SphI

Double Digest