Negative controls in cloning? - (Aug/16/2005 )

I use the double-digest method to cut the PCR product and the plasmid. Then I will do the ligation. So, what kinds of negative controls should be used in the ligation steps?

Somebody told me as the following, but I can not understand. so, waht kind of negative control do you usually uased?

Negative Controls:
Reaction without insert -> if colonies then backbone is not completely double
cut
Reaction without insert and ligase -> if colonies then backbone is not completely single
cut
Reaction without template and ligase -> if colonies then insert is contaminated with PCR
template

Those are the three major controls, but we usually only use the first one.

If it helps you understand, think about what's in your ligation reaction, where each component came from, and what the possible contaminants are. Positive colonies will arise from any plasmid containing a gene for drug resistance. These plasmids can be one of three things.

1) Your ligated construct - your insert ligates to your vector as you expect, giving rise to a drug resistant bacteria, and therefore a colony.

2) Self-ligated vector without insert - the cut vector ligates its two ends together without an insert. This can happen if you're working with blunt-end ligation, your sticky ends have partial overlap in sequence complementarity, or if there are trace amounts of contaminating nuclease that cause your vector's ends to become blunt. This type of potential contaminant is the reason the "no insert" control is run. The control also addresses the possibility that your vector wasn't fully cut - if you don't actually remove the small piece of DNA between your cloning sites, the vector will be a linear piece of DNA with complementary ends. You can minimize the problem by treating vector (NOT the insert) with phosphatase prior to gel extraction.

3) "Source" vectors - in a ligation reaction, you combine two different pieces of DNA with complementary ends. Each of those pieces of DNA came from somewhere - the vector from a full-length plasmid and the insert from either a full-length plasmid or a PCR amplification of genomic DNA or a plasmid. Whatever the case, those source materials are present until you remove them, which is usually accomplished by agarose gel extraction - you separate your components based on size and specifically extract the productive ligation material away from the source material. However, if your source DNA runs at similar positions to your ligation material, you may wind up co-purifying them, and they'll get included in your transformation. This is why the "no ligase" controls are done - to make sure that the purified ligation materials aren't carrying any source vectors.

so, what kind of negative controls do I really need in my experiment? please tell me the detail cocktail, thank you!

hi
i do this negative control : mix ligase bufer water and enzyme divide it in two and add in the first one plasmid + insert and in the second one plasmid + water.
fred