Agarose gel for very small DNA fragments - (Aug/23/2006 )

Hi,

Since you can't do a dissociation curve with TaqMan i need to run a gel to confirm that my Realtime PCR was specific and gave me my fragment of interest.

I have two product that i need to check- they are 63bp and 69bp in lenght.

What % gel do i use to see such a small fragment???
Also what voltage and how long should i run the gel for??


I usually make a 1.5% gel - (100mls of 1 XTAE + 1.5g of routine agarose) and run for 40 mins at 80 volts. I stain in EtBr after i run the gel.

However, this does not really give me a clear photo.

I can distinguish the positive from the NTC but the postive band no very sharp.

Any suggestions on how i can inprove my gel????

Cheers

You could try a 6 or 7 % gel, I have used 7% for 30 bp, worked good. Just use special agarose for high-percentage gels, otherwise it won't cook up right and get clear.

I have used TBE as buffer and let it run at 90 V for 45 minutes. worked fine.

You could also use polyacrylamid gels, 6%, in TBE, if you do not have this special agarose but PAA.

Good luck!

this is a little different...I would use a lower percentage, say around 2, and run it much more slowly, perhaps over 2.5-3 hours

you could try more than one trick and see which one works well for you?

A colleague used to make 4% gel with a special agarose (Neusieve) to c different bands of 12 bps.

Nusieve agarose give better resolution.
A 4 to 5% gel will be ok for cooking.
I recommend to add Bet in the gel and stain to ensure a good stain overall as 60pb doesn't take good the stain due to small size.

hi Pops
I used precast gels from invitrogen to check 20 and 30mers,
works for me

good luck

For analysing the DNA ladder of TRAP assay which range anywhere from ~46bp and above with 6bp increment on a 10% native polyacrylamide gel. I use 0.5X TBE and run at 100V for ~2 hrs. If garose does not give good resolution you can try this out.