So, I have been cloning a lot of genes over the years in various plant transformation and overexpression vectors and even though its a struggle always to generate positive clones, I usually am able to confirm the sequences and use them for expression studies.
I am now cloning a truncated plant TF in the Tobacco Rattle virus (TRV2) system after confirming its clone in PgemT Easy cloning vector and have been failing from the past three months. I have used to sites NcoI and Xho1 for directional cloning, the Promega rapid ligation system and have kept for ligation at various time intervals ranging from short to overnight ligation, using minimum 50ng vector to 150 ng in concentration at a ratio of 1:1,2:1,3:1 and 5:1. However, on using TRV2 specific primers I am only getting amplification of the vector at about 150bp instead of getting about 450bp amplification (insert+vector). Please suggest me few tips.
I think that you could generate this effect if either one of your enzymes was not cutting then the vector can re ligate to give the short amplimer but insertion cannot take place. If you have not already done so try single cutting with each enzyme separately to check the integrity of your enzymes
Paul Rutland Respected sir, I have been using double digest enzymes in a single buffer (Thermo Fischer) conventional enzymes and digesting overnight and have been getting a single band after electrophoresis . I will re-digest them as you have suggested,one after the other. Thank you so much for the suggestion.
Probably best to cut half with Nco first and half with Xho first just to make sure that both enzymes do cut as once the first enzyme has cut it will be difficult to see if the second has also cut.
Bipasha Bhattacharjee Try these
1. A Fresh ligation buffer and use it from aliquots
1:1 ligation must work in this case
2. Heat the v\digested vector and insert prior to putting ligase and buffer
3. If you are gel extracting the fragments, it might be possible that the kit is not removing salts and carryover
4. Include controls in your transformation; transform the digested vector only
Bipasha Bhattacharjee 100-150ng vector is sufficient, provided that vector must be linearised completely.
Hi Bipasha,
Please first ensure that your vector is properly digested or not from both the enzymes? In that case, Add each component in your ligation reaction except the insert. This will serve as the control. If you are getting more colonies in insert free ligation control then your vector is not properly digested with one of the enzymes. I recommend for sequential digestion.
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