5 replies to this topic

[epigenetics]

Hello,

I need some advice, which universal primer (M13 or T3/T7) i should send with my sample for sequencing.
How to determine this? I am doing TOPO TA cloning using TOP10 cells from invitrogen and pCR®4-TOP vector.
This vector looks like:

Seq
201 M13 Reverse priming site
240 T3 priming site
290 PCR product
340 T7 priming site
360 M13 Forward priming site

Thanks,

Edited by epigenetics, 13 April 2009 - 07:05 AM.

[mdfenko]

i would use m13 primers. that way your pcr product sequence will be in the cleanest part of the data.

[hanming86]

T3 and T7 probably would work too because it's like 50bp apart . normally u can't read the first roughly 20 bp of sequencing.

[mdfenko]

hanming86, on Apr 14 2009, 07:13 AM, said:

T3 and T7 probably would work too because it's like 50bp apart . normally u can't read the first roughly 20 bp of sequencing.

but it really cleans up after ~100 bases.

[epigenetics]

Thanks to all of you.

i am using invitrogen TOPO cloning kit, But not getting as much as colony i want. I guess, i need to optimize conditions.
After PCR, I do Gel extraction of my band using qiagen kit, then i add Taq pol, Taq Buffer, dATP to get A overhang and keep it @ 72 degree for 10 minutes, then i do cloning using pcr4 TOP vector using 4 microlt of my sample, 1 microlt of vector, and 1 microlt of salt (according to invitrogen's ).
Then i use 2 microlt of this for transformation using TOP 10 cells, and proceed further.

What might be wrong? My insert is 246 bp (DNA from human skin), plasmid DNA is like 4 KB.

Thanks,

[jiajia1987]

epigenetics, on Apr 15 2009, 03:54 AM, said:

Thanks to all of you.

i am using invitrogen TOPO cloning kit, But not getting as much as colony i want. I guess, i need to optimize conditions.
After PCR, I do Gel extraction of my band using qiagen kit, then i add Taq pol, Taq Buffer, dATP to get A overhang and keep it @ 72 degree for 10 minutes, then i do cloning using pcr4 TOP vector using 4 microlt of my sample, 1 microlt of vector, and 1 microlt of salt (according to invitrogen's ).
Then i use 2 microlt of this for transformation using TOP 10 cells, and proceed further.

What might be wrong? My insert is 246 bp (DNA from human skin), plasmid DNA is like 4 KB.

Thanks,

".............then i add Taq pol, Taq Buffer, dATP to get A overhang and keep it @ 72 degree for 10 minutes, then i do cloning using pcr4 TOP vector....."

you need to clean up your PCR products before cloning as the NH4 in the PCR buffer can kill the cells.