Improving Transformation Efficiencies
From Roel Funke, summarizing responses to a query to bionet.chlamydomonas September 1995
rfunke@exelixis.com
Following are some of the responses I got in answer to my question about transformation, and whether or not plasmids should be linearized before using them to transform Chlamydomonas. Thank you.
Stefan Fabry was curious to hear what got back to me, so I will pass on his angle:
We are currently also trying some tricks to improve transformation (low-methylated DNA) and will be curious whether you got some additional tips from the community.
From Karen Kindle
I find slightly higher rates of transformation for linearized plasmid DNA (see Kindle, PNAS 87:1228) though it’s not dramatic. The higher rates are probably due to cutting outside of the selectable marker rather than having integration sites be “random” on the plasmid and sometimes disrupting the marker. Linearization is absolutely critical if you want to do insertional disruption followed by plasmid rescue, but I think that if you’re trying to complement a mutant it wouldn’t be so important. I believe that someone in Joel Rosenbaum’s lab tried transformation through the cell cycle with synchronized cells. We have found that the most important factor is how long the cells have been in liquid culture; there are also some strain to strain differences. We continue to get transformation rates between 10-4 and 10-5, usually about 1000 independent transformants per plate using 8 x 106 cells.
From Lisa Ellis
ellis@wustlb.wustl.edu
It is clear that by using linearized DNA you reduce the chances that your insertion will be accompanied by a large deletion in the nearby genomic DNA. This is very important if you are tagging a gene and want to clone out the wild type gene by cloning DNA adjacent to the site of insertion. In addition, linearized DNA tends to be less rearranged when it goes in. I’m not sure if anyone really knows if linearizing gives better transformation rates, although it seems possible that ends are more recombinogenic than circular molecules. Also, if you use PEG, make fresh solutions with some regularity to maintain high transformation rates.
From Sally A. Adler
techne+@andrew.cmu.edu
As to the question of plasmid linearization: It makes a large difference. Linearize. I use Xmn I to open all amp^r plasmids (it’s in the middle of amp^r). I like to stay as far away from my insert as possible. It’s a very well behaved, not too expensive enzyme that is AT-rich enough to be rare in chlamy genes. You can get it from New England Biolabs.