Difference between pgemt vectors and pcambia vectors
Answers
Explanation:
As with all cloning experiments, doing controls will help you understand whether your experiment is working as expected. Doing ligations with vector alone and of vector plus control insert (I think the pGEM-T kit provides template and primers for a control) are a good start. For TA cloning, one problem can be that after long term storage or multiple freeze-thaw cycles, the plasmid can lose the terminal T overhangs. When this happens, the frequency of recircularized vector increases - and this can be seen as an increased number of colonies in a self-ligated vector control. This background can mean you need to screen (many) more colonies to get ones with insert.
As far as the size of plasmids is concerned, unless you are digesting the plasmid DNA, I don't think you can gain much from trying to estimate plasmid size from agel of supercoiled DNA. There are several factors that cause problems. First, supercoiled DNA doesn't run predictably alongside linear markers (although you can buy supercoiled markers); second, nicks and cuts in supercoiled DNA can give rise to open circular (non-supercoiled circles) and linear plasmid molecules that tend to run more slowly than supercoil; third, many of the high copy-number vectors (like pGEM) can exist as multimers in the cell, which can produce bands of double, triple the size of the original vector. Thus having a number of bands in one lane doesn't indicate muliple plasmids - the bands may just be different forms of the same plasmid.
For normal TA (and other) clonings my group routinely use chemically competent JM109 from Promega (as supplied with the pGEM-T cloning kit). For more difficult cloninings - including very large inserts I sometimes use electrocompetent DH5alpha.