Alternative to RACE-PCR

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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magicsiew
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Post by magicsiew » Tue Jan 18, 2011 2:19 pm

Hi JackBean, I have looked through the protocol of the hybridization method. My sample genome size is very huge, if I were to build the library, I doubt how many hundreds, or thousands of plate I will require to make before getting the library done. In this case, it doesn't seem like a good alternative. Anyway, thank you for your information.

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JackBean
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Post by JackBean » Tue Jan 18, 2011 2:22 pm

well, you can have several different vectors for different insert sizes. How large is your genome?
There is formula, which tells you, how many colonies you need to check to have like 95% chance to find your gene. I don't remember it, but I will check it out, when I come home.


OK, it this wouldn't work, what about some specific purification of your say mRNA?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Tue Jan 18, 2011 2:28 pm

Hi JackBean, I have looked through the protocol of the hybridization method. My sample genome size is very huge, if I were to build the library, I doubt how many hundreds, or thousands of plate I will require to make before getting the library done. In this case, it doesn't seem like a good alternative. Anyway, thank you for your information.

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JackBean
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Post by JackBean » Tue Jan 18, 2011 3:47 pm

your plant has genome size ~550 Mbp, rigth?
The formula is N = ln(1-P)/ln(1-n), where P is the probability, you will find your gene and n is the ratio of genome size to insert size.
Maybe you could start with larger inserts at first, e.g. with BACs or cosmids.

Unfortunatelly, you would have to check plenty of plaques, but that's how it worked. And if you still don't have anything better, it's probably the best way. Unless you're going to sequence your plant ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Tue Jan 18, 2011 5:36 pm

Hi, I am surprised, how do you know my plant genome size is ~550Mbp? Yes, it is. Thank you again for your information :)

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canalon
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Post by canalon » Wed Jan 19, 2011 1:49 am

To make things a bit easier:
Make a digestion with a relatively (1 or more) rare cutter and run a PFGE. Use hybridization to identify the band that contains your favorite gene and clone in a cosmid. Then sequence that or repeat with a more frequent cutter and shorter fragments.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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JackBean
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Post by JackBean » Wed Jan 19, 2011 8:58 am

I remembred, what plant are you working with, so I looked up on the internet :-P
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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