NanoBio: Primer Design

Primer Design

You will need 4 primers per knockout that you design.

2 will be for the PCR to clone out the antibiotic resistance. These are 70 bases long.
The other 2 are for verifying knockouts with colony PCR. These are 25 bases long.

This is the trickier set. Be careful! I will use NapC from E. coli as an example here.

First, find the sequence of your gene(s) including 50 bases upstream and 50 bases downstream.

NapC is on the antisense strand, so this sequence is the reverse complement of the coding sequence. It doesn't matter which way you look at it though! Do whatever is easiest for you to look at.
The start and stop codon below are in lower case.
NapC is 603bp

CGCTCACAAAGTAACTCTCTGGCTTCAAGCATACCCACGCAATAACCCTG

ttaAAAACCTGGCTCGACTTCACGCATATCGGGCAGCTTGTGCGCTATCCCTTTATGGCAATCAATACAGG

TTTGCCCATCTTTCACCGCCTGGTCATGCATCTTCGCGGCAACCGATTTCTGGGCGGTTGTATCCATATAC

TCGAAGTTGTGACAGTTACGGCACTCCTGCGAGTTATTGTCCTTCATGCGCCGCCACTCATTCTGTGCCAT

CGTCAGACGATGAGCTTCAAATTTCTGCGGCGTGTCAATAACGCCAAAAATTTTACCATACAGCTCTTTAC

TTGCTTTGAGCTTGCGTATCATCTTCGGCACAAACTCGTGCGGAACGTGACAATCCGGACAGGTCGCACG

GACGCCGCTACGGTTGTTGTAGTGCACGGAATCCATGTATTCCTGATACACCGTGTTGCGCATTTCGTGG

CAGCTAATGCAGAACTCTTCGGTATTGGCTTTTTCCATCCCGGTGTTAAAGCCACCCCAGAAGACGATGC

CGCCAACAAAACCGATCAACAGCAGCGTCCCCAGCGCCAGACGGCTGGGGGTACGCCACCATTTCCAC

AGGCGCTTAATCAGACCAGGCTTACGGTCAGAATTTCCCATAATAACCTCTTATTTCCCGTAACCTTTTGATGGGGTAAAGGTATTCCcat

AATAACCTCTTATTTCCCGTAACCTTTTGATGGGGTAAAGGTATTCCCCA

This is the easier set! Just copy and paste the fist 25 bases of each of the above primers, and you're done!

--Heather M. Jensen 14:52, 9 June 2009 (EDT)

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