Hello,
I did a ligation using pCDNA3.1 (5.4 kb) and a gene of interest(1.1kb). I transformed and plated DH5alpha cells with the following: pCDNA3.1+gene, pCDNA3.1 linearized, pCDNA3.1 linearized+ligase, DH5alpha alone. Most colonies observed on plasmid+insert plates, few in pCDNA3.1 linearized with or without ligase, and none in the cells alone plate.
After two digestions of 8 colony DNA using EcorI and HindIII from NEB and Promega respectively, I get the image attached. I am rerunning with an undigested plasmid this time since I forgot.
My question is, how can the gene be present in the last two samples without a plasmid? how did the cells grow without the resistance of the plasmid? and how can I be sure that the digests worked and that is why i dont have the gene of interest in the first 6 lanes?
Thank you for your help!
You can also try colony PCR using the gene specific primer.
Good luck.
To help more fully some more information is required:
1. What are the sizes on the ladder used. These would be important for identifying if you have just the vector or a linearized backbone and vector.
2. What conditions are you using for your digest, is it a double digest - if so are both those enzymes from different providers compatible in the same buffer.
3. How are you handling the DNA from extraction to gel running - any precipitation steps or anything like that?
4. Can you should that both enzymes function individually, set up a non digested, single digest with EcoRI and a single digest with HinDIII.
If you can answer all these then a clearer understanding will be possible.
It may well be that the cloning failed because you only got single digesting of you vector and therefore it ligated back together with better efficiency in the vector + insert experiment.
Simon Cass: Thank you for your response.
The ladder used was Tri-Dye 2Log ladder, so the higher bands correspond to the plasmid and the lower ones to the insert.
The two digests were done separately due to incompatibility of the buffers from both companies.
The DNA was acquired with a 5mL culture and isolated by miniprep from Monarch Plasmid Miniprep kit.
I ran the samples after the first digest with only HindIII, and the gel looked exactly the same (first 6 lanes had the bands at the same size as plasmid and the last two lanes had a band at the same size as insert) Which makes me think that the last two colonies had only the insert ligated onto itself. But how did they cells grow on that plate without the plasmid?
Thank you for your help
Hi Olga,
Ok so yes I agree the higher band in the first six lanes appears to just be your vector.
I would advise checking both you enzymes work separately (Why are you not using both NEB enzymes in CutSmart?), and comparing that to your perceived 'clones'.
I doubt that there has been any ligation in the last two lanes, this is likely some artifact of DNA that is either a plasmid that has been modified in the bacteria or some other excised DNA from the host genome. In the former case retention of the ampr gene would allow growth. There is always the possibility of escaper mutations rendering ampicillin ineffective too.
If you cant see a clear vector and insert in the diagnostic digest, I advice starting again, and checking at each stage everything is working.
Good luck!
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