false positives when cloning microsatellite fragments - please help! - (Aug/04/2006 )
Hello,
I am having problems cloning a range of fragments (from approx. 200-900 bp in length) as part of microsatellite library construction. On most occasions, when using the TOPO cloning kit, I find a high percentage (60-100%) of false positives (white colonies, but no apparent inserts when amplified with M13 primers). However, this is not always the case - on 3 occasions I have had 100% success.
From reading other people's messages on this website, I understand that this problem could be due to an imperfect vector to insert ratio. I can see how it is possible to calculate the desirable ratio when you just want to clone one band, but when there is a smear of products, I am less sure how to go about this calculation. From looking back at my pre-cloning gels, I do not see a marked difference in concentration of products between those that resulted in a 100% successful cloning reaction and those that resulted in a high percentage of false positives.
Any help will be much appreciated,
Thank you,
Katharine
When making a library, your small fragments will have a tendency to saturate your ligations. This is due to the fact that the smaller fragments are in greater concentration when determining the 'smear' concentration by mass (ie. ng/ul).
One way to avoid an over abundance of small fragments and false positives is to split your cloning efforts by fragment size. Use a pool of fragments that are large, a pool that is midsize and a pool that is small. You should see an increasing number of false positives with the smaller library, but you can focus on optimizing that library while your larger fragment libraries are more or less consistent.
Hi Vasussci,
Thanks very much for your reply. I will do as you suggest by gel purifying different size fractions of the smear.
Thanks again,
Katharine
One way to avoid an over abundance of small fragments and false positives is to split your cloning efforts by fragment size. Use a pool of fragments that are large, a pool that is midsize and a pool that is small. You should see an increasing number of false positives with the smaller library, but you can focus on optimizing that library while your larger fragment libraries are more or less consistent.
Last week I set up 2 cloning reactions: the first I cloned the usual smear of fragments associated with m'sat library development, the second I gel purified a range of fragments from about 500-600 bp. The next day, as usual, there were lots of colonies. I did the M13 colony PCR on 8 colonies from each cloning reaction. None of the 8 from the first cloning reaction worked and only one of the 8 from the second cloning reaction yielded a product. I tried diluting the template for 3 of the PCRs (x10, x100 and x1000 diltuions), but to no avail. I've run out of ideas! Does anyone have any further advice??
Thanks very much for your reply. I will do as you suggest by gel purifying different size fractions of the smear.
Thanks again,
Katharine
When making a library, your small fragments will have a tendency to saturate your ligations. This is due to the fact that the smaller fragments are in greater concentration when determining the 'smear' concentration by mass (ie. ng/ul).
One way to avoid an over abundance of small fragments and false positives is to split your cloning efforts by fragment size. Use a pool of fragments that are large, a pool that is midsize and a pool that is small. You should see an increasing number of false positives with the smaller library, but you can focus on optimizing that library while your larger fragment libraries are more or less consistent.