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Thankyou for all your suggestions.
As a final option, i scanned for different NaCl concentrations at which the Protein can show binding to Ni NTA.
The Ni NTA to His interaction is so specific that it remains at such high concentrations of NaCl like 1M or even 2M.
The protein that initially creared problem in not binding to any column, bound very well to Ni NTA itself at 1M NaCl in buffer.
I suggest sincerely, if someone has problem in Ni NTA (His tag binding problem) to try higher salt concentrations before concluding any thing.
#10354 His tag protein purification problem
Posted
on 26 January 2005 - 07:30 AM
#125202 Need help for PCR for AT rich gene
Posted
on 10 December 2011 - 11:05 AM
Often it is suggested to use betaine to make a PCR work in AT-rich regions. Also the companies say this about their additives (Q-solution (= betaine), PCRx Enhancer Solution etc). No idea if it's true, but an attempt might be useful.
#125196 Need help for PCR for AT rich gene
Posted
on 10 December 2011 - 08:06 AM
I have done PCR through poly AT regions as long as 30 bp. You probably read the Su paper, and indeed the extension temperature is the key. In my case, I could not get the PCR to work with an extension temperature higher than about 63 degrees. I doubled the normal extension time, but that was probably more than necessary. You might try out some of the newer enzymes such as Phusion that have DNA binding motifs to hold the enzyme on the strand.
