I'm trying to perform ligation by cleaning up the insert and vector prior to ligating them. Concentration is coming down to very low after the cleanup which is not giving good results. Pls help me .
You can often increase the yield of column purifications by adding hot (70c) water or elution buffer to elute the cleaned up dna and leave it on the column for twice the recommended time. Then if you are precipitating the dna to increase the concentration add a co-precipitant like 1ul of 20mg/ml glycogen which will bring down more of the dissolved dna
If you start with more molecules, then you end up with more molecules. Try a larger volume & concentration of the vector & insert.
If possible, don't do a cleanup step. If your enzyme is heat-inactivated that means you can skip the cleaning. If the insert is a PCR product & it's a single, bright band on the gel, then just use the PCR directly in the ligation reaction & skip the cleanup.
Are you phosphatase treating the vector? It will make a huge difference in reducing the self-ligated vectors.
you can co precipitate the insert (PCR product) with the vector then recover the dried DNA pellet directly in 1x ligation buffer....
Katie A S Burnette Didier Poncet Paul Rutland thank u :) I'm using Bbs1 to digest the vector which cannot be heat inactivated. and my inserts are gRNAs annealed in annealing buffer. I'm purifying both for which the concentration is very less after purification.
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