PCR of lambda gt10

chihara chihara at alm.usfca.edu
Mon Jul 31 18:22:04 EST 1995


I have a student who used the following procedure, worked fine. She 
subcloned the product and sequenced from there. Take a plug of a well 
isolated plaque, dissolve in 100 microlit. of water.  vortex. She used a 
Mg free cocktail prepared by mixing 5 microliters of above template 
stardog at u.washington.edu (Kevin Lease) wrote:
>Could someone please send me a good protocol for PCR with a lambda gt10
>clone as my template DNA?  I am particulary interested in hearing how
>people begin their PCR to denature the capsid.
>
>Thanks,
>        Kevin Lease
>
with 1 microliter of forward and reverse primers at a conc. of 25 pico 
moles/microliter, 1 microliter of the mixture of dNTP's (10 mM) 5 
microliters Tzq buffer (10X) 2.5 mMoles. 0.5 microliters Tzq enz. and 
water to 50 microliters.
Thus she seems to have used the phage directly depending on the burst to 
provide the DNA.?? If this doesn't make sense, I'll push her to see if 
she left something out. 
Hope this helps.





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