probelm with Full-length cDNA library construction - (Aug/14/2006 )
i use SMART kit to construct Full-length cDNA library, but there are some problems. i wanna konw how to increase my full-length cDNA in my library, and what method could increase the library titer.
Are you having difficulty:
1) amplifying your library?
2) cloning your library?
or
3) screening your library?
There are a lot of options depending on where you're having the most difficulty.
1) amplifying your library?
2) cloning your library?
or
3) screening your library?
There are a lot of options depending on where you're having the most difficulty.
i need to screen some soybean lipid and protein genes in my library. my material is soybean inmature seed. we got EST and Full-length cDNA by 5'sequence. but there is a question in my experiments. The full-length cDNA in my library was little . i just want to konw how can i get more full-length cDNA. Thank you!
When you say your full-length cDNA are 'little', do you mean they are short in length or you just don't have that much to work with?
You can remedy this situation by fractioning your library into short and long elements.
For example, If you do a random amplification you get something that looks like a smear on a gel. If you purified from this gel, instead of using the whole smear for the library, split the gel gragments into small medium and large. This way the smaller fragments do not dominate subsequent ligations into your vector.
No matter what strategy you use for cloning you will need to make several libraries or pool several amplifications to saturate the number of unique genes you can find with that strategy.
You can remedy this situation by fractioning your library into short and long elements.
For example, If you do a random amplification you get something that looks like a smear on a gel. If you purified from this gel, instead of using the whole smear for the library, split the gel gragments into small medium and large. This way the smaller fragments do not dominate subsequent ligations into your vector.
No matter what strategy you use for cloning you will need to make several libraries or pool several amplifications to saturate the number of unique genes you can find with that strategy.
Thanks to vasussci .
Now i prepare to fraction my dscDNA into two parts including 500-1500bp and >1500bp by agal electrophoresis . But there are a new problem exist when i using LD-PCR syntheses dscDNA. And i give you a pic , please give some advices .
I think you're ok. If you have the two different fractions they'll amplify differently, but you should still see a smear (as shown in the gel on the left). Be careful not to preferentially amplify the smaller fragments, by increasing your extension time.