Hi Veera, there's just one protocol - if this doesn't work something else is wrong:

- 4.5µl amplified product directly taken out of the PCR reaction (freezing lowers efficiency)

- 0.5µl pENTR/D-TOPO

- 1.0µl salt solution from the kit (either pure for chem.trafo or 1:4 for electroporation)

- incubate for 15 to 30min at room temperature on the bench

- directly do the transformation into E. coli (TOP 10 preferred)

If this won't take you the whole next day counting all the positive colonies you should check if:

- the PCR product gives a strong signal on an agarose gel

- you have chosen the right antibiotic (Kan)

- the preincubation time of your cells with the plasmid is between 30 and 60 min

- your cells are competent at all (provided plasmid in the kit)

- the TOPO vector is not spoiled (this can happen, use control reactions from the kit to check)

In 10% of all integration events the insert goes in the other way around, if your product shows a certain kind of composition at the 3'end (yes, not only GGTG but also others) it sometimes happens that 90% of all inserts go in the wrong way.

Greetings to the north and happy 20th anniversary! Cheers, Robert

Hi Veera, there's just one protocol - if this doesn't work something else is wrong:

- 4.5µl amplified product directly taken out of the PCR reaction (freezing lowers efficiency)

- 0.5µl pENTR/D-TOPO

- 1.0µl salt solution from the kit (either pure for chem.trafo or 1:4 for electroporation)

- incubate for 15 to 30min at room temperature on the bench

- directly do the transformation into E. coli (TOP 10 preferred)

If this won't take you the whole next day counting all the positive colonies you should check if:

- the PCR product gives a strong signal on an agarose gel

- you have chosen the right antibiotic (Kan)

- the preincubation time of your cells with the plasmid is between 30 and 60 min

- your cells are competent at all (provided plasmid in the kit)

- the TOPO vector is not spoiled (this can happen, use control reactions from the kit to check)

In 10% of all integration events the insert goes in the other way around, if your product shows a certain kind of composition at the 3'end (yes, not only GGTG but also others) it sometimes happens that 90% of all inserts go in the wrong way.

Greetings to the north and happy 20th anniversary! Cheers, Robert

HI Veera, Robert's answer is excellent. I have been using Invitrogen Topo cloning since the early days. I would add that if you have very good pipettes and tips you can get away with even smaller reactions (this is especially true for all the subsequent LR reactions you will do - always scale these down - you only need 2 ul to transform). I would add that we have found gel purification of your PCR reaction always works much better than using total PCR products.

Because this is a Kanamycin based plasmid you will need to let the bacteria produce the drug resistance marker before plating (Kan disables translation) so let them 'recover' for >30 minutes prior to plating on plates.