11 replies to this topic

[hbn]

I would like to amplify DNA using T7 DNA polymerase, using a primer which has a T7 promoter sequence in front of it.
This means the primer will anneal to the template, but the promoter sequence will not.
Does anyone have any idea if this will work? In other words; does T7 DNA polymerase need double stranded promoter sequence?
Please advise!

[blin100]

Summary: yes it needs double stranded. Simply PCR your DNA first using a hi-fi polymerase with your T7/Sp6 primers to get double stranded product with the promoter site there. (Convenient way of getting both sense and anti-sense probes for ISH)
Please see source for confirmation:
http://www.invitroge...nscription.html

[hbn]

blin100,
Thanks for your reply! I thought of that too, but unfortunately, PCR is really difficult in this region, so I'm afraid that will not work...
But thanks for your help anyway!

[blin100]

that is most unfortunate. Is it a repeat heavy region? Those are the worst
If you can deal with a bit of extra RNA at the end, you could topo clone it into PCR2.1, the default topo plasmid which has a T7 site downstream of the A insertion point.
http://tools.invitro...2_1topo_map.pdf

[hbn]

Jup, highly repetitive It's almost impossible to make PCR primers...
But I'm starting from total genomic DNA and I want to perform target enrichment, so I somehow have to select for the region of interest... I guess it's impossible

[blin100]

Ah, that is terrible. is this for ISH?

[hbn]

No for next-gen sequencing...

[blin100]

ahh, makes more sense. ISH has been on my mind recently and I saw T7
I'm just thinking out loud here, but what if you made biotinylated complimentary oligos to the repeats, conjugate them to biotin beads or resin to basically make a selection column? Run your sheared dna through it, wash and elute?

[hbn]

I guess you are doing ISH?
It's quite a repetitive genome (plants are difficult to work with ) so making specific probes may also be a challenge.
But still it's not that bad of an idea... Do you have any idea how long the probes and the sheared fragments have to be?

[blin100]

Yea, I wish I were doing ISH...that worked
Aha plants unfortunately, I have absolutely no idea hahaha, just an idea that popped into my head. I would assume its possible since people do poly-A selection columns, you should be able to just substitute your oligo for the oligodT in those columns. I would guess they wouldn't be that long? maybe 20 bp?

[hbn]

Haha, so you are TRYING to do ISH? But not on plants, I assume?
Yes, plants are sometimes terrible to work with... I read some papers that this region was highly repetitive but it's worse than I expected! :|
Yes, a lot of target-enrichment kits work this way; with a probe and than 'fishing' out the probe-bound DNA sequences...

[blin100]

I'm doing ISH on adult mouse olfactory epithelium, its a wonderful system but RNA is super sticky there, so basically you get background regardless of how you wash

Are there any benefits to working on plants?
I'm feeling like c elegans is the best model to work with, if only they had humanoid organs....

Is next gen sequencing of this super necessary for your work? It seems do-able but just barely