[PCR] why the template band was missing?

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bbs_r
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[PCR] why the template band was missing?

Post by bbs_r » Wed Mar 29, 2006 9:15 am

I run 2 electrophoresis, the first one with library cDNA alone. The second one was a PCR with the same recipe I shown in the other post.

I am curious that why the template band was missing? (although it is not important to my experiment, I just wondering why sometimes the template band was there and sometimes don't)

Thank you very much again!
Attachments
lib_proof.JPG
Library cDNA alone (2% agarose gel electrophoresis)
lib_proof.JPG (3.27 KiB) Viewed 1301 times
15.JPG
PCR (2% agarose gel electrophoresis)
==============
Ladder --- A --- B
==============
A = Practical
B = Control
15.JPG (5.31 KiB) Viewed 1301 times
Last edited by bbs_r on Wed Mar 29, 2006 3:26 pm, edited 1 time in total.

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canalon
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Post by canalon » Wed Mar 29, 2006 12:45 pm

How much did you load in each lane?
In your PCR, according to your post, your template was 5ul in 250ul that you may not load entirely... How much in your Library cDNA alone?
Patrick

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

bbs_r
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Post by bbs_r » Wed Mar 29, 2006 3:23 pm

You are right, the amount of DNA loaded alone is 10 uL. While the PCR one only loaded < 5 uL (estimate).

May I concluded that "the template band in the PCR picture is not at a detectable level rather than it is missing from the gel"?

Thank you!

bbs_r
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Post by bbs_r » Wed Mar 29, 2006 3:28 pm

By the way, I just have one more question:

why does the control band (PCR) shown up like two thick bands sticking together? While sometimes I only got one clear band...

Just a bit confused.

Thank you!

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