4 replies to this topic

[Mehjabeen]

My Masters thesis topic is to clone cry1a gene into TA vector then subclone into pgex6p2. My insert size is 1815bp TA vector 3900bp. Even if i get pcr product but colony pcr gives no desired band.My thesis submission is next month & am really worried & frustrated. Please, any suggestions will be welcome

[Andrea Fortina]

Check your competent cells and your PCR products again. Use different enzymes to check you products, and make sure you get the right products.

[bob1]

If you can also give some details of how you are going about things, that will help. 

 

Some things to think about for now: Are you using Taq polymerase? If not, you need to incubate your PCR product for 15 min with some Taq and some dATP to generate the A overhang, as most other polymerases don't generate overhangs. for TA cloning I generally use a proof-reading polymerase to generate the product, then do the Taq incubation. Note you might need to purify the product before the Taq incubation to ensure the buffers are right for Taq.

 

Colony PCR is notoriously tricky as products from the bacteria can inhibit the reaction. Do you have a positive control that is also from a colony (i.e. not purified DNA)? One solution to this is to pick the colony (get the smallest amount you can manage), dip the colony into 100 ul of ultra-pure water, then use 1 ul of this for the PCR. You may need to dilute further also.

[labtastic]

One solution to this is to pick the colony (get the smallest amount you can manage), dip the colony into 100 ul of ultra-pure water, then use 1 ul of this for the PCR. You may need to dilute further also.

 

This is essential to get colony PCR to work reliably. Unless people do this, I don't believe them when they say "the colony pcr says the insert isn't there".

[827753]

 

One solution to this is to pick the colony (get the smallest amount you can manage), dip the colony into 100 ul of ultra-pure water, then use 1 ul of this for the PCR. You may need to dilute further also.

 

This is essential to get colony PCR to work reliably. Unless people do this, I don't believe them when they say "the colony pcr says the insert isn't there".

 

The lab I work in uses screening primers sufficiently away from the cloning site that even a recircularized vector will generate a visible PCR product.  If this fails then something else is wrong.