PCR in 96-Well Plate - (Mar/02/2007 )

Hello everyone

I want to perform PCR on 200 gDNA samples so I thought to use 96-well plate. Can someone provide me of some useful and practical advice on how this can be done? Where should I keep the mastermix in order to be able to load it with a multi-channel pipette on the plate? Is the multi-pipette acurate enough to load the precise volumes which are needed in the PCR reaction? Please give me some advice!

I've been doing a lot of this lately. Have you got a multi-dispenser pipette like an eppendorf Multipette? Far, far better than a multichannel for this. Otherwise, load 8 mastermix aliquots into each of the 12 wells in the top row, then take single aliquots into each row with the multichannel. It's not too bad.

You'll find the last few wells might be foamy and under-volume, no matter what kind of pipette you use. But for PCR mastermix it's not a problem. I've successfully done a PCR with only 10-15ul mastermix in the last few wells or tubes (because of pipette inaccuracy or I just miscalculated) and it's fine.

Try 2 rows of a PCR plate before you do the full 96 wells. You never know, maybe your lids don't sit properly and the PCR will leak, or something. (this happened to me)

Are you going to be running all 96 samples on a gel? That'll be fun

Also, if you're screening 200 samples for the presence of a certain gene, you can combine groups of (say) 10 samples together, forming 20 new template mixes. Screening those 20 samples is much easier than screening 200, plus you save reagents. Then when you find some positive groups, perform individual PCR on the original samples.
That's what we've been doing, but it might not suit your situation.

I've been doing a lot of this lately. Have you got a multi-dispenser pipette like an eppendorf Multipette? Far, far better than a multichannel for this. Otherwise, load 8 mastermix aliquots into each of the 12 wells in the top row, then take single aliquots into each row with the multichannel. It's not too bad.

You'll find the last few wells might be foamy and under-volume, no matter what kind of pipette you use. But for PCR mastermix it's not a problem. I've successfully done a PCR with only 10-15ul mastermix in the last few wells or tubes (because of pipette inaccuracy or I just miscalculated) and it's fine.

Try 2 rows of a PCR plate before you do the full 96 wells. You never know, maybe your lids don't sit properly and the PCR will leak, or something. (this happened to me)

Are you going to be running all 96 samples on a gel? That'll be fun

Also, if you're screening 200 samples for the presence of a certain gene, you can combine groups of (say) 10 samples together, forming 20 new template mixes. Screening those 20 samples is much easier than screening 200, plus you save reagents. Then when you find some positive groups, perform individual PCR on the original samples.
That's what we've been doing, but it might not suit your situation.

QUOTE (Zouden @ Mar 2 2007, 05:50 AM)

I've been doing a lot of this lately. Have you got a multi-dispenser pipette like an eppendorf Multipette? Far, far better than a multichannel for this. Otherwise, load 8 mastermix aliquots into each of the 12 wells in the top row, then take single aliquots into each row with the multichannel. It's not too bad.

You'll find the last few wells might be foamy and under-volume, no matter what kind of pipette you use. But for PCR mastermix it's not a problem. I've successfully done a PCR with only 10-15ul mastermix in the last few wells or tubes (because of pipette inaccuracy or I just miscalculated) and it's fine.

Try 2 rows of a PCR plate before you do the full 96 wells. You never know, maybe your lids don't sit properly and the PCR will leak, or something. (this happened to me)

Are you going to be running all 96 samples on a gel? That'll be fun

Also, if you're screening 200 samples for the presence of a certain gene, you can combine groups of (say) 10 samples together, forming 20 new template mixes. Screening those 20 samples is much easier than screening 200, plus you save reagents. Then when you find some positive groups, perform individual PCR on the original samples.
That's what we've been doing, but it might not suit your situation.

Thanks a lot! I sure won't run 96 samples on the same gel ! I have an Eppendorf multipipette in the lab. How about loading the template? Can I trust a repeater pipette? Is it acurrate ? I will not run 96 samples at once, but should I laod water in the wells that remain empty? Too many questions I know but I would appreciate your help !!!!

Repeater pipettes are not as accurate as a multichannel, but they're much faster and (as I said) it'll be accurate enough for dispensing mastermix.

I used to be very exact with my PCR volumes, but now I've realised it doesn't matter that much for most work (as long as the mastermix is still consistent!) and I no longer add water to my negative controls to compensate for lack of template.

The amount of template you add is important. If you have a multichannel pipette that can do 1ul accurately then use that. It's better to use too much than to risk not having enough in some of your wells. But don't stress too much about having identical volumes of template because your gDNA extractions probably have wildly difference concentrations (if they're like mine )