Hello, all.
I've been unsuccessful in cloning a 6.3 kb gene into pGEM T easy.
Are there other plasmids that are optimal for cloning an insert of this size? Alternatively, is there a protocol that is usually followed for cloning larger inserts?
I’ve been successful in cloning smaller inserts (3 kb or less). I’ve even successfully cloned a 2kb fragment of this 6.3 kb gene into pGEM T easy. These successful attempts were all verified by sequencing.
I'll describe the steps I've taken:
The 6.3 kb gene is PCR amplified using the AccuPrime high fidelity Taq DNA Polymerase. I've checked the company’s documentation and confirmed that this polymerase adds a single deoxyadenosine to the 3’ ends of the PCR product, which facilitates TA cloning into pGEM T easy.
After PCR amplifying, the products are run on a gel and the size of the band is confirmed by comparing against a lambda bste II ladder.
The ~6.3 kb bands are cut out of the gel, pooled, and gel extracted using the Qiagen gel extraction kit. I’ve done this entire process multiple times, and my yield after gel extraction is usually ~25 ng/ul (final volume is 30 ul).
Next the ligation is performed with varying molar ratios of insert:vector. This is the equation used to calculate the amount of insert to add to achieve the various molar ratios:
📷
[(ng of vector x kb size of insert)/kb size of vector] x insert:vector molar ratio = ng of insert
The kb size of the vector is 3 kb (pGEM T easy). The kb size of the insert is 6.3 kb. I’ve tested 1:1, 3:1, 6:1, 9:1, 12:1, and 1:3 insert to vector ratios. Following this overnight ligation at 4 degrees Celsius using T4 DNA ligase from NEB, electrocompetent cells are transformed using electroporation, plated on carbenicillin plates, and colonies are screened via colony PCR (and sequencing) to determine whether or not the insert is present.
I’ve tested over 100 colonies over the multiple iterations of this process, and they’ve all been empty. I don’t think that the issue is the ligase per se because I was able to ligate in a 2 kb fragment of this same gene using the same ligase.
Dear Christopher,
pGEM-series plasmids, as all pUC-derived vectors based on derepressed ColE1 replicon, is extremely high copy. When you transform your cells and plate them, those having an empty vector or no vector at all easily outgrow these you want to obtain. The gene you tried to clone was pretty big for such high copy vectors, although I saw even 10 kb inserts in pCR4-TOPO (the same replicon).
So, If I were you, I would try this:
1. Shorten the recovery time after shocking the cells (either chemical or electroporation, and better use the latter, it works much better for larger plasmids), so that the cells have no opportunity to divide
If the above fails
2. Find the TOPO cloning kit (you can buy it off ThermoFisher), TOPO ligation is much more efficient than standard ligase-driven reaction, plus pCR vectors work better with larger inserts
And as the last resort
3. Use lower-copy plasmid, such as pACYC184 or pBR322 (the latter is also high copy, but somewhat lower than pUC), but you will need to prepare it yourself for TA-cloning.
Good luck and best,
Marcin Golebiewski
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