T/A cloning

[Michelle.Gleeson at SMTPGWY.AGRIC.NSW.GOV.AU]

     Hi everyone,
        We use Promega's pGEMT-vector here in Australia, with moderate 
     success.  We had experienced a drop off in efficiency in several of 
     our kits over time.  Technical support said that the ligase buffer 
     supplied should be warmed gently (no more than 37C for a few minutes) 
     to dissolve the precipitate which we often find, even in new batches.  
     We were also told the buffer is unstable to freeze-thaw cycles, so we 
     now thaw once and aliquot.  They also said not to use any other ligase 
     than that supplied with each kit - they apparently optimise the 
     activity to give the best results.  Since the activity of pGEMT kits 
     here is so much reduced over activity people have seen in when 
     visiting the US, these things are important to us.  BTW, what do 
     people in the US and UK pay for the kit? Here it is AU$145 for the kit 
     without competent cells.