RealTime-PCR Question

[Amrazzz]

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Didn't know where to post it, and I need desperate help here.

I have approximately 40 samples and I don't want to run regular PCR to do the gel. I've already done Real time PCR with SYBR green and wondering if I can run this amplified product on the gel? Also is loading dye necessary?

Thanks!

 

[Web MD]

Go for it. Use loading dye

 

[ohgodidonteven]

How can you tell how far your DNA has run without the dye? What's your normal indicator for the bands? EtBr or some other alternative?

 

[bold text]

Your sample will float out of the wells if you don't use loading dye.

 

[ohgodidonteven]

Your sample will float out of the wells if you don't use loading dye

There they will comingle and evolve like primordial cells in a great sea, using energy from the electricity running through the box to become more complex until they are able to escape their buffery confines, eager to seek out new genetic material to consume and assimilate. It happened to a friend of mine.

 

[Amrazzz]

How can you tell how far your DNA has run without the dye? What's your normal indicator for the bands? EtBr or some other alternative?

I use a 1000bp DNA ladder as a reference. We usually use EtBr gels to visualize under a GelDoc once the DNA is tagged with a loading dye. Just wondering if this was necessary if we use SYBR green which is fluorescent.

 

[DrYoda]

There they will comingle and evolve like primordial cells in a great sea, using energy from the electricity running through the box to become more complex until they are able to escape their buffery confines, eager to seek out new genetic material to consume and assimilate. It happened to a friend of mine.

 

[ohgodidonteven]

The one thing you might want to watch out for is since the SYBR Green binds to the DNA much like EtBr does (and is probably more effective), you will find your bands will not be as bright as usual (assuming they show up).

 

[runrunrudolph1]

As previously stated, your dye has cellulose which will bind to your sample and allow it to fall to the bottom of the well. Without it... your sample will be floating around in the buffer. Also.. use a ladder to compare it to your sample.

 

[ohgodidonteven]

Hmm, I was told it was glycerol. Yes, find your protocol and follow it. Also, ask your PI.

 

[Amrazzz]

Hmm, I was told it was glycerol. Yes, find your protocol and follow it. Also, ask your PI.

Problem is, PI and post doc are out of town for the week, so its up to me to get this done.

Anyways, I found a protocol, and hopefully it will work. Thanks for the help ya'll!

 

[Web MD]

You owe us

 

[iPodtosis]

I don't understand the logic here.

If you are trying to get quantitative information, analysis of the RT-PCR with SYBR green should give you more accurate data than running it on the gel.

If you are trying to see the band size, then why do you use the expensive SYBR green at the first place? normal PCR would work just fine.

If you are trying to get both information, then maybe ya you would do 1 PCR, but as someone mentioned above, SYBR green binds to DNA, and it could change the fluorescence, and migration distance.

To answer your question, ya loading dye is necessary.

 

[Amrazzz]

I don't understand the logic here.

If you are trying to get quantitative information, analysis of the RT-PCR with SYBR green should give you more accurate data than running it on the gel.

If you are trying to see the band size, then why do you use the expensive SYBR green at the first place? normal PCR would work just fine.

If you are trying to get both information, then maybe ya you would do 1 PCR, but as someone mentioned above, SYBR green binds to DNA, and it could change the fluorescence, and migration distance.

To answer your question, ya loading dye is necessary.

I know... We had primer dimers in some of the samples after doing the MeltCurve. Plus I'm looking at a knocked out gene and the effect on its inhibitor. The RT-PCR amplified for the knockout when it shouldn't have so I want to run a gel and see if it was something else that's interfering. Also, gels can tell the approximate size of the target gene, which is something which I also need to make sure.

 

[Tedster]

I often run out the products of a real-time PCR assay on an agarose gel w/ EtBr to see if the amplicons are the correct size if its the first time I have tested out a new assay. I have even used the gel to purify and sequence the products. So should be fine. I have not noticed any shift in the gel from Sybr.

That being said- you might want to use a higher resolution ladder as most RT-PCR amplicon sizes are around 100bp due to efficiency parameters. For sure at least use a 1kb+ ladder vs the standard 1kb.

 

[iheartmints]

Hmm, I was told it was glycerol. Yes, find your protocol and follow it. Also, ask your PI.

Yeah I'm almost certain it's the glycerol in the loading dye that keeps your samples from floating out from the wells

 

[ShinyDome19]

1) Glycerol is used since it is much more dense compared to water which is the base ingredient in typical running buffers...It does not bind up your DNA, I can see that as being very counter productive.

2) You can run RT-PCR amplified samples on an agarose gel - there are no major differences b/w RT-PCR and regular old generic PCR aside from the indicator dye (in your case SYBR Green) and having a PCR machine that can measure it.

Out of curiosity, why wouldnt your PCR amplify your knockout? Typically, atleast in my experience, a knock out is procured by inserting a nonsense strand or a mutation into the middle of a target gene..not removing the gene altogether:

(Wild Type)
[Promoter]----------[Terminus]

(Mutant/Knockout)
[Promoter]-----[Inserted Nonsense]-----[Terminus]

So, hypothetically, if your forward primer targets your promoter region and your reverse primer targets your Terminal region, you'll get a product either way...just a differnt size.

 

[Amrazzz]

1) Glycerol is used since it is much more dense compared to water which is the base ingredient in typical running buffers...It does not bind up your DNA, I can see that as being very counter productive.

2) You can run RT-PCR amplified samples on an agarose gel - there are no major differences b/w RT-PCR and regular old generic PCR aside from the indicator dye (in your case SYBR Green) and having a PCR machine that can measure it.

Out of curiosity, why wouldnt your PCR amplify your knockout? Typically, atleast in my experience, a knock out is procured by inserting a nonsense strand or a mutation into the middle of a target gene..not removing the gene altogether:

(Wild Type)
[Promoter]----------[Terminus]

(Mutant/Knockout)
[Promoter]-----[Inserted Nonsense]-----[Terminus]

So, hypothetically, if your forward primer targets your promoter region and your reverse primer targets your Terminal region, you'll get a product either way...just a differnt size.

Its very possible to knockout the gene all together without just adding a nonsense sequence in there. The way my mice were KOed was flanking the gene with loxP sites in embryonic stem cells and transplanting them into a blastocyst. We then bred these "floxed" mice with a mouse positive for Cre recombinase. The loxP sites are an indicator to Cre recombinase which then "snip" out the gene. The leftover sequence after the gene is removed is just the loxP termini. I want to make sure that the primers aren't amplifying the loxP sites instead.

Thanks for the info on the dye! Running gel at the moment