Stable Tandem Repeats?

Scott scj-13 at home.com
Thu Aug 23 10:56:15 EST 2001


> Assuming that you overcome the problem, please post what
> works back to this newsgroup.

OK, I've done a little bit of experimentation with one of my constructs 
and thought I would report back to the group with my findings.

The insert in question is about 2.8 Kb and is composed of four 700 bp 
direct repeats. I was building up this construct in Novagen's NovaBlue 
host. Once I got to these 4 copies I was seeing fairly substantial 
expansion and contraction of this insert. Once I posed my original 
question to the group I got several responses. Most suggested STBL2 from 
BRL or SURE from Stratagene. I also found Novagen had a strain(BLR) 
designed for this purpose as well. I acquired all three strains and did 
a side by side comparison. I transformed each with the original miniprep 
from the cloning of the insert containg the 4 repeats. This was the most 
homogenou DNA prep I had given that it had been through the fewest 
generations. I performed all incubations at 30?C since there is some 
evidence that the lower temperature also reduces recombination. In fact, 
the BRL STBL2 strain specifically states to grow it at 30?C when it 
contains a troublesome construct. The day following the transformation I 
picked 4 colonies from each plate and restreaked them for miniprep 
analysis. When I came in today they had all grown up, but to varying 
degrees. This is all very subjective and fairly hard to explain, so 
please bear with me. The SURE cells looked pretty unhealthy. Kind of 
blotchy I guess. The STBL2 strain looked normal while the BLRs were very 
robust. I performed minipreps on all 12 cultures and cut out the inserts 
using sites in the vector, and ran a gel. All 12 transformants had the 
2.8 Kb insert and I couldn't see any signs of smaller inserts as a 
result of recombination. I'm sorry this may not really help other in 
chosing one of these strains over the others, however, they were all 
substantially better than the simply recA1 NovaBlues. I'm not sure if my 
situation of only a few large repeats will react differently than those 
constructs which contain many very small repeated sequences. My best 
advice is what others have given me, you'll just have to try a few out. 
For now I will probably continue to use the STBL2 strain as it's gotten 
favorable reviews from others and appears to be working well for me. One 
caveat is the 30?C incubations. I would much prefer the faster growth 
rate 37?C provides and will probably try that out and see if it has an 
impact. Any ideas? If you have any questions or suggestions please let 
me know. Thanks to everyone for their help.

-Scott




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