running itme for gels - (Oct/06/2006 )

Hi,

I have Taqman PCR products that i need to run on a gel to comfirm that thay are the correct size- the amplicons vary between 50- 150bp.
I have Matephor agarose to help resolve these small fragments.

But have a few questions-

1. Should i use TAE or TBE?
2. What % gel should i use?
3. How long shoud i run the gel for and at what volts?

Thanks

bbeing so small products, I would use a 2% gel.
I never figured out what's the difference between TAE and TBE in terms of running, so I can't help there.

I usually run at 100 V, which seems to be fine for all fragments. Your fragments are small, so don't run it too long; no more than 10-15min. If you run it at 50V, then they go slow, obviously. But again, 15 minutes should do.
Needless to say, don't forget your 100bp marker


quote name='Pops' date='Oct 6 2006, 01:06 PM' post='72041']
Hi,

I have Taqman PCR products that i need to run on a gel to comfirm that thay are the correct size- the amplicons vary between 50- 150bp.
I have Matephor agarose to help resolve these small fragments.

But have a few questions-

1. Should i use TAE or TBE?
2. What % gel should i use?
3. How long shoud i run the gel for and at what volts?

Thanks
[/quote]

For historical reasons, TAE is the most common used buffer. However, its buffering capavity is rather low, and it tends to become exhausted during extended electrophoresis. (the anode becomes alkaline, the cathode acidic). Replacement of the buffer or recirculation between the two eservoirs is therefore advisable when carrying out electrophoresis for long periods of time at high current. TPE and TBE (Tris-phosphate and Tris-borate buffer) have higher buffering capacity. Double stranded linear DNA fragments migrate approximately 10% faster thru TAE than through TBE or TPE. Still, the resolving powers of these systems are almost identical, with the exception that the rsolution of supercoiled DNAs is better in TAE than in TBE. --> Maniatis

I run metaphor gels at 82 V for 80 minutes. I would use a 3% gel if I really needed resolution at 50 bp. I use TAE and stain with Sybrsafe, but it probably makes little difference.