6 replies to this topic

[Epigeneticist]

Invitrogen TOPO TA Cloning - 505bp insert.

Problem:
1. Using the vector for LaZ/blue white screening - I'm getting many blue colonies with a few white colonies and a majority do not contain my insert.
2. Using the vector with the lethal gene,  I'm getting a few large white colonies (positive clones) with many small white colonies (false positives and they are not satellite colonies).

Things I've considered and ruled out as a problem:
1. Insert lacking A overhangs - I've used fresh PCR product, gel purified product and I've tried 10,15 and 20min for my final extension along with using a 1.5min extension during cycling.
2. Molar Ratio: I've tried vector:insert ratios of 1:1, 1:3, 1:5. Along with using just 0.5, 1 or 2ul of fresh PCR product (gel shows single band).
3. LB Kan plate: antibiotic is good

I ran the control with the kit - generate control PCR product (750bp), ligate and transform = many blue colonies and few white positive clones. => low efficiency.


LAST TROUBLESHOOTING IDEA - POSSIBLE SELF LIGATION OF VECTOR

1. Ligation Step, is it critical to add components of ligation mixture in a certain order and kept at a certain temperature? Add salt and insert then vector? Adding salt and vector first at RT may allow for self ligation before insert is added?
2. Should this mix be keep on ice while preparing until everything is added then place at RT for incubation?
3. How much to mix ligation? Very small volume and hard to tap side of tube, pipette up and down to mix?

Thanks

[bob1]

Which of the topo TA kits did you use?

How did you prepare your PCR product (specifically which polymerase did you use)?


1)The ingredients should be added in the order suggested in the manual.
2)Yes, keep on ice.
3)As much as you can, use a 2 ul or 10 ul pipette and it should work.

[Epigeneticist]

Bob1

I've tried Invitrogen TOPO TA Cloning Kits - TOPO TA Cloning for Sequencing and TOPO TA Cloning. Procedure for ligation is transformation is identical. Pretty much similar kits, just different vectors - first vector has lethal gene and no need for blue white screening, and second kit uses vector with LacZ for blue/white screening.

I've used two different Taq Polymerases
1. Fermentas Taq
2. Zymo Research Taq - better for bisulfite converted DNA and suitable for TA cloning too

PCR reaction is 50ul with 40 cycles and 20min final extension

Package insert does not state which order to add the ligation components or what temperature to store mix while preparing it, just to incubate at RT after mixing together. The volumes of components are listed in this order - PCR product, salt solution, water and vector. Does not mention to add them in this order and does not list them numerical order either. This should not be this finicky. I'm just concerned that the vector could self ligate at warmer temperatures if not mixed properly with insert or if allowed to sit at RT without insert added yet.

Thanks

[bob1]

Epigeneticist, on 20 January 2013 - 12:26 PM, said:

Bob1

I've tried Invitrogen TOPO TA Cloning Kits - TOPO TA Cloning for Sequencing and TOPO TA Cloning. Procedure for ligation is transformation is identical. Pretty much similar kits, just different vectors - first vector has lethal gene and no need for blue white screening, and second kit uses vector with LacZ for blue/white screening.

OK, just checking that you had actually used a Kan vector. Some of the kits used to have different vectors to the ones used now.

Quote

I've used two different Taq Polymerases
1. Fermentas Taq
2. Zymo Research Taq - better for bisulfite converted DNA and suitable for TA cloning too

PCR reaction is 50ul with 40 cycles and 20min final extension

This should be fine, making sure you weren't using one of the proof-reading mixes which remove the A overhang.

Quote

Package insert does not state which order to add the ligation components or what temperature to store mix while preparing it, just to incubate at RT after mixing together. The volumes of components are listed in this order - PCR product, salt solution, water and vector. Does not mention to add them in this order and does not list them numerical order either. This should not be this finicky. I'm just concerned that the vector could self ligate at warmer temperatures if not mixed properly with insert or if allowed to sit at RT without insert added yet.

Thanks

Ah, but it does, as you listed above...  The vector should not self ligate as it has two T overhangs.  If the kit has not been stored properly the topoisomerase conjugated onto the ends of the vector and/or the ATP in the salt solution will degrade quickly, which will mean that the kit will not work well.

As a note - you surely only need one clone with the proper insert, so if you have one already, why the fuss?

[Epigeneticist]

Bob1

Reason for the fuss - I need 10 positive clones from each of my two inserts for bisulfite sequencing. I would like the cloning to be more efficient so I can get all my clones from the first reaction without having to repeat it.

So, if the kits are stored properly as they are, what could cause self ligation? Adding salt then vector and letting this sit at RT for a brief period of time before adding insert? Could this really happen? Maybe I'll let the tube sit on ice then assemble everything in the order mentioned to keep the mixture cold before RT incubation.

[bob1]

Epigeneticist, on 20 January 2013 - 01:27 PM, said:

Bob1

Reason for the fuss - I need 10 positive clones from each of my two inserts for bisulfite sequencing. I would like the cloning to be more efficient so I can get all my clones from the first reaction without having to repeat it.

So, if the kits are stored properly as they are, what could cause self ligation? Adding salt then vector and letting this sit at RT for a brief period of time before adding insert? Could this really happen? Maybe I'll let the tube sit on ice then assemble everything in the order mentioned to keep the mixture cold before RT incubation.

Fair enough.  Potentially the salt could cause self ligation, though it seems unlikely to me.  Have you tried the Invitrogen help pages/tech support?

[Epigeneticist]

Emailed tech support and I'm waiting for a response. Also, I have downloaded/read their troubleshooting publication along with the troubleshooting guide in the back of the package insert. I feel as though I have exhausted my resources. I'll try the cloning again after I hear back from Invitrogen.