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Dear Steve,
Thank you for your help.
Is the three step Secondary PCR preferable for my project ?
Or should I stay with the two steps with longer primers?
Thank you,
Tami
Dear all,
I am trying to clone a 3000 bp insert into a 5000 bp plasmid .
Primary PCR has hardly worked, but I got 400 ng of mega primers for the secondary PCR .
Secondary PCR did not work (2 step ) , I used 5 min extention time. 1:20 primer/insert ratio.
I transformed into One Shot TOP10 Competent Cells, 5 lamda of DpnI treated PCR mix.
Positive control worked for the transformation, DpnI treatment of the parental plasmid worked, as I only got one colony.
I will be happy if you could help me improve my Primary PCR yield and help me with the Secondary PCR.
Thank you,
Tamar
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