I am using NotI and NdeI to digest a 750 bp insert in a 5400 bp vector backbone. I am hardly getting any colonies after transformation. Once I got plenty of satellite colonies in 3:1 ligation ratio plate and a few decent looking colonies in the 5:1 plate (using Top10 E.Coli competent cells); however, after performing plasmid isolation from the 3:1 plate, the plasmid concentration was found to be really low (
I do not completely understand what you are doing, but if you are cloning, did you try to purify insert and vector from before ligation? it should show you whether the digestion was complete and also improve the following ligation.
I agree with the comments given above.
1. You need to check compatibility of the enzymes and buffers. If one enzyme inhibits the other one, you need to perform digestion in a batch. Inactivate the enzyme used first prior to the second batch of digestion.
2. Quality control (QC) of your target materials after each step is very critical. You need to purify, characterize and quantify cut DNA (insert and vector). Use appropriate controls.
3. Did you purify your ligation products? Did you use chromatographic/column-based techniques or ethanol precipitation to purify your ligation reaction products? if ethanol precipitation is used for purification, you need to air-dry the DNA pellet prior to resuspension of the pellet.
4. Use a nuclease-free water to elute/resuspend your target DNA during purification.
5. Think of the factors affecting efficiency of transformation
6. Use the right antibiotic with the optimum concentration
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