advice re: shRNA vector sequencing - (Jan/25/2008 )

Hello,

I've just cloned my shRNA into an expression vector (Clonetech's pSiren Retro Q Green). I am now looking to get my clones sequenced. Does any one have any helpful hints on how to get this done? Is there anything special that I need to ask my sequencing facility of choice to do? I ask because one of my labmates could not get his hairpin sequenced by one facility (I think I'll use a different facility but how will Iknow which one is good??).

Many thanks.

Good question, since I have the same trouble

Tell the sequencing people the problem and ask them for suggestions. Our sequencing people use DMSO in their reactions. This usually helps.

i did my sequencies with Amersham big dye kit.
in a pcr equipment using this program :


95° 20’’
50° 15’’
60° 60’’
Hold 4°
30 cycles

then add

15µl water
2µl Na Ac1.5M pH 5.2 (also 3M is fine too)
120µl EtOH 95%

Transfert in eppi 1.5ml
Spin 12000rpm RT 30’
wash with 100µl 95% EtOH
dry 15' on the bench without heating.

all my shRNAs were sequenced by that method. Not 100% success but i'm pretty confident for 95% success

Thank you Fred and Scolix,

I've spoken to the sequencing technitians (good idea! Why didn't I think of that before?) and they have an alternative protocol for hairpins involving DMSO and maybe higher temperatures. I'm keeping my fingers crossed.