Easiest cloning strategy for deleting part of plasmid using adapters? - (Oct/25/2008 )

Hi,

We have a plasmid encoding GFP with a mitochondrial targeting sequence (MTS). We want to make a plasmid with the MTS removed. The MTS is flanked by two unique sites (NheI & BamHI) . The whole protein (including MTS) is flanked by unique sites (NheI & NotI) . It seems there are three ways to remove the MTS:

1) PCR the GFP only, with primers that generate Nhe1 and Not1 sites, then paste the Nhe1 and Not1 cut PCR product into the Nhe1 and Not 1 cut and gel purified plasmid.

2) Cut the plasmid with NheI & BamHI to cut out the MTS, dephosphorylate the plasmid, then religate the plasmid using small phosphorylated adapters and ligase.

3) Cut the plasmid with NheI & BamHI to cut out the MTS, gel purify the plasmid, then blunt the ends with Klenow and religate the plasmid with ligase (blunt end ligation)

Option 2 is the most appealing, since it seems the most direct and can keep the enzyme sites. However, I have not been able to find any guidelines for doing this!

• How long should the adapters be?
• Will this work with adapters that are only 8 bases long?
• Is there risk of concactamers, even if the adapters are not self complementary at all?
• Is the correct protocol to simply cut the plasmid with NheI & BamHI, dephosphorylate the plasmid, phosphorylate the adapters, mix the adapters and plasmid (including the dephosphorylated MTS sequence) and add ligase?

Nice post Jon. All three methods are fine. To answer your questions on option 2, as far as i know, there are no minimum or maximum number of bases to include in an adapter as long as the Tm of the adapter is strong enough to stay annealed at the temperature you are going to use it at - i.e. room temperature for ligation. I should qualify my answer by saying that i have a lot of experience in cloning but little experience with adapters.

There are two things i would consider when designing my own adapters: 1) you want the oligos to be long enough or more specifically, unique enough so that when they anneal to each other they anneal efficiently, stay annealed, and do not want to anneal to themselves; 2) there are enough bases between the two restriction sites so that if you want to use both restriction sites in a single cloning in the future you can - if they are too close they may not have enough flanking bases after one of the sites is cut for the other site to cut efficiently. Note: you also have the opportunity to add additional restriction sites not already present in your vector/insert to your MCS by designing them in your adapter.

8 bp is probably ok but it really depends on the Tm. If you're not sure, add a couple more bases - it will increase the Tm and can't do any harm - might cost you an extra $5.

Yes, there is risk of concatamers but there is always a risk of concatamers. The secret to preventing this is using a sensible amount of adapter in your ligation. If you just try to plough your adapter into the vector, then your molar ratio will be massive because the adapter is so small and you increase the chance of concatamers - small insert = lots of moles. So calculate it out or if you're feeling lazy do a couple of 10-fold dilutions and you should be fine (there isn't a lot of science in this).

That protocol is fine. Just a couple of things: 1) i assume you were already going to do this, but if you weren't, electrophorese the vector after you've cut it to remove the insert you don't want anymore; 2) phosphorylate the adapter as single-stranded oligos rather than the double-stranded adapter (think you were going to do this). Phosphorylation is more efficient on single-stranded DNA.

Good luck,

Rob


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