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How do I insert a DNA sequence into a multiple cloning site and add restriction

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#1 Lee Zelaya

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Posted 17 October 2011 - 06:42 PM

I'm are trying to insert the DNA sequence below into the multiple cloning site of a plasmid that has been digested with BamHI (cuts 5’GGATCC3’) and EcoRI (cuts 5’GAATTC3’). Design PCR primers to amplify the sequence highlighted below, and incorporate the appropriate restriction sites. Assume that a 6-nucleotide long sequence will be sufficient for high specificity amplification (in reality, at least 15 nucleotides are required).


5’-5'GATTACGGTAAATGCCGGATTAACCCGGGTTATCAGGCCACGTACAACTGGAGTCC-3’
3’-3'CTAATGCCATTTACGGCCTAATTGGGCCCAATAGTCCGGTGCATGTTGACCTCAGG-5’

#2 sks1

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Posted 21 October 2011 - 05:34 AM

u sud also add at least 3-5 more basepairs to 5' end before RE site so that Restriction Enzyme can sit properly over DNA and cut it efficiently..

gd luck




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