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Old 08-10-2017, 09:40 AM   #1
kirupha
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Location: Gainesville

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Default How to determine the size of the insert in the vector.

Hi all,

I am working with PCC1BAC vector which contains my insert. I have sent the BAC DNA to sequencing to determine the size of the insert. I have got back the sequencing results and using serial cloner software I'm trying to determine the insert size. Can anyone help me with this.

Thanks,
Kirupha
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Old 08-10-2017, 09:57 AM   #2
Olaf Blue
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The pCC1BAC vectors are all 8,128 bases no matter the linearizing nuclease:

http://www.epibio.com/tech-support/d...s/pcc1bac_hind

Otherwise digest the clone with the enzyme NotI as it cuts ~300 bases either side of the insertion site. Will generate a ~7.5 kb vector band and at least one insert band depending on the insert sequence and NotI recognition sites. Run on a 0.7-1.0% agarose gel, stain, photograph, measure migration using a ruler and suitable sizing program.
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