3
votes
Can someone link me to resources on the efficiency of sticky end ligation?
The best resource for troubleshooting ligations I found (and use frequently) is this NEB page. That is assuming you've already referred to the instructions provided with the enzyme you're using. In ...
- 103
2
votes
Can Bacteria Repair Dephosphorylated Plasmids?
As per WYSIWYG's suggestion, I tried the digest and dephosphorylation again with SalI and ran a gel. The gel showed that SalI was less than ideal at cutting this plasmid.
As you can see, the most ...
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2
votes
Accepted
Freezing after restriction digest
From my experience I would say that it is no problem to freeze the digested DNA, when the enzyme is inactivated (even when not, for this time period I wouldn't expect much damage).
However, if ...
- 51.1k
1
vote
Separate DNA fragments with very different size (oligo and lambda-DNA)
A spin column might work for your purposes since you can do multiple rounds of binding if your volume is too large to bind in a single pass, but for DNA of that size a lot will be irreversibly stuck ...
- 5,513
1
vote
Ligation without purifying insert
Thanks all for your comments guys. I tried purifying using PCR purification kit. I could recover 120 ng of purified fragment from 500 ng. It is ok for my further work.
- 630
1
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What happens after the purification step in Hi-C sequencing?
So, after the restriction digestion, the sample consists of DNA fragments with 5' overhangs. These are filled in by Klenow DNA polymerase, adding biotin-dCTP to both ends of each fragment. Then the ...
- 17.6k
1
vote
Can someone link me to resources on the efficiency of sticky end ligation?
To measure the frequency of indels at the ligation site you can use a vector with a unique restriction site in the lacZ gene. With a colorimetric assay you can count the number of white cfu. Perfect ...
- 3,432
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