I use Lithium acetate transformation method for transforming intact circular plasmid into yeast.
In past, I always aliquot a new salmon sperm DNA from commercial tube, then boil in water for 10 min, and put on ice until use for transform, and get a lot of transformants. (and never used re-bolied salmon sperm DNA)
But for these two time that I get very little of transformants, I use a re-bolied sheared salmon sperm DNA that have a lot of "freez - thraw - boiled" cycle for many time because I aliquot so much and cannot use all. Thus, I kept the boiled DNA in freezer, when I want to use I will boil it agian and agian like a cycle of freez - boli
Then, my assumption is the salmon sperm that have many time of freez - thraw - boiled cycle will give a low transformants efficiency?
Does any one face the case like me? or any suggest, please provide
Hi there.. We have found that you can reboil carrier DNA but it does reduce the transformation efficiency in a negative fashion... Reboiling your carrier DNA can have a dramatic effect on Transformation efficiency so I only worry about it when i am doing a screen that requires 10e6 transformants. Other times the reboiling effect is minimal... I believe it has to do with the state of your carrier DNA (fragment size) Years ago we found the larger carrier DNA works better for transformation than smaller sized carrier. As the size of your carrier DNA decreases so does your transformation efficiency and yield. This may explain the reboiling effect. As the size of the carrier is reduced by what ever treatment the transformation efficiency and yield are reduced.
I rather found out that if once in a while re-boiling the salmon sperm DNA might actually help increase the efficiency. But yes if you are frequently freeze-thawing you vial..then it may lead to diminished transformation efficiency.
Hi, as far as I remember there is a limit of freeze-thawing recommended. It should be in Gietz protocol or on Sigma Aldrich datasheet for salmon sperm. It might be around 4 times...It was long ago I used it...but yes, for sure after some number your efficiency should drop, it was tested. so make small aliquotes:)
I suggest you buy some fresh, sheared SS DNA from Sigma and compare. In our case (embarrassingly) we once had a case where a new arrival in lab had made Li-chloride instead of Li-acetate and that surely reduced transformation efficiency.
I've used the same vial of SS DNA over several freeze/thaws. Sometimes, I will reboil the vial. I had some difficulties with transformations recently. Turns out that my water bath wasn't holding temperature. One degree made a difference, apparently.
Dear Saxena, Dashko, Kerscher, Lee-Soety,
Thank you for your help.
I will try to get my transformant efficiency back to the high level.
As Sofia said the Gietz TRAFO protocol mentions this issue, see the links attached. Specifically it is advised to boil up to 3-4 times and that it is not necessary to boil the carrier DNA every time. I hope this helps.
http://home.cc.umanitoba.ca/~gietz/method.html
Article Transformation of yeast by lithium acetate/single-stranded c...
Hi there.. We have found that you can reboil carrier DNA but it does reduce the transformation efficiency in a negative fashion... Reboiling your carrier DNA can have a dramatic effect on Transformation efficiency so I only worry about it when i am doing a screen that requires 10e6 transformants. Other times the reboiling effect is minimal... I believe it has to do with the state of your carrier DNA (fragment size) Years ago we found the larger carrier DNA works better for transformation than smaller sized carrier. As the size of your carrier DNA decreases so does your transformation efficiency and yield. This may explain the reboiling effect. As the size of the carrier is reduced by what ever treatment the transformation efficiency and yield are reduced.
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