help in cloning 6kb pcr fragment into topo? - (Oct/09/2007 )

Does anybody have any experience in cloning a 6Kb PCR fragments into TA TOPO or blunt TOPO?
I've been trying for 2 weeks and all i get are empty vectors.

I used 30-70ng (4ul) of my PCR fragment for the TOPO reaction
1ul salt buffer
1ul Topo vector

tried incubation at room temp for 10mins and 30mins
tried transforming 2ul to all 6ul of reaction with the TOP10 competent cells @42degrees for 45sec - 1min

picked abt 10 white colonies and did a restriction digest to check for my insert. only see the vector band.
Did sequencing to check, and true enough, all empty vectors.

any help or suggestion is appreciated

-uginz-

Are you sure that you have the A-overhang needed for topocloning?

I have no experience from the plasmids you mension so I am maybe not the right person to answer...

I am using TOPO XL, that is for larger inserts (3-10kb). Maybe that can be worth to try instead?

My cloning reaction (for the XL vector) is performed in 5 minutes in room temperature.

I have always 30s at 42 degrees, with all vectors I have used.

-Ammie-

thanks!
I'll suggest this kit to my supervisor.

my pcr product is supposed to have a 3'-A since Taq was used in the polymerase mix

-uginz-

Fist, are you purifying the PCR product?
Some protocols say it’s not necessary, but if you don’t do it, you could have lot of background.
Is it a new kit?? When this kits are old and/or frizz-warm many times, they start to give lot of background.
Be sure you are amplifying with Taq for T vectors or
amplifying with Pfu for blunt vectors.

I prefer to use cloning vectors with a toxic gene (this way, most of the colonies have insert and X-gal/IPTG are not necessary) in stead of LacZ based vectors.

Ej:
- Zero Background™ Cloning (Invitrogen)
- topo XL PCR cloning kit (Invitrogen)
- GeneJet PCR cloning kit (Fermentas) CHEAPER!

-aztecan princess-

QUOTE

Fist, are you purifying the PCR product?

yup, gel purified

QUOTE

Is it a new kit?? When this kits are old and/or frizz-warm many times, they start to give lot of background.

yup, brand new kits.

QUOTE

Be sure you are amplifying with Taq for T vectors or amplifying with Pfu for blunt vectors.

it should have 3'-As since i'm using Adv2 polymerase mix which is supposed to have Taq.

QUOTE

I prefer to use cloning vectors with a toxic gene (this way, most of the colonies have insert and X-gal/IPTG are not necessary) in stead of LacZ based vectors.

great idea. I assume it's the Zero background cloning Kit?

-uginz-

It suppose there are zero background.
Some times you have one or two false positive from 10 colonies.

-aztecan princess-