Molecular Biology PCR

[ALewo90]

Hi there

I need some help with a PCR experiment I had to do for an assignment and I'm not sure if I completely understand whats happening in the agarose gel photo. I've added the gel photo and also the molecular weight marker used.

 

lanes (from left to right) 1=negative control, 2=chicken DNA, 3=cauliflower DNA, 4= Molecular weight marker

 

this experiment was to see if using the same primers produced from cauliflower beta-actin, and by adding them to chicken DNA if they would amplify the same sequence.

 

I would expect it not to amplify the same, and the gel photo shows the brightest chicken fragment higher than the cauliflower. but then there is also a fragment that is at the same height as the cauliflower but not nearly as bright.

 

I'm still trying to wrap my head around all this and not sure about what i'm actually seeing, if someone could help me on the right path that would be great!

 

Cheers

Ashlee

[Genecks]

I'm trying to follow your logic.

It appears that you have developed a Southern Blot. You have four lanes. Each lane is discussed, as you have previously mentioned.

I'm stuck at this next part. Let me see if I understand.

Someone has added the DNA for Cauliflower beta-actin into the Chicken DNA. I am not sure how the methodology went about this.

Was this a cell that had DNA of chicken and cauliflower in it?

Was the second lane from an extract of a Chicken DNA, which then had the primers that are later translated and transcribed in cauliflower cells in order to make cauliflower beta-actin, separately loaded with the Chicken DNA, thus causing the second lane to have a mixture of Chicken DNA and cauliflower beta-actin primers?

Another thing not mentioned is the molecular weight of various things you expect to see.

If the cauliflower primers bound to the chicken DNA, then yes, you're going to see a more amplified spot higher up on the molecular marker spot. If when cutting the hybrid chicken/cauliflower DNA with Hae III, it will then cut a the ends with the chicken and cauliflower DNA, then yes, you're going to get a higher molecular weight.

I'm not sure if I'm seeing things right here, though. Perhaps another member can chime in.

[CharonY]

Why do you think that this is a Southern?

The simplest explanation is unspecific amplificates. Note that the size is fairly low (in the low 200). If the run was not stringent enough there can be some products there. Note that there is something even lower, which are likely primer dimers (depending on their length).

Of course there is also always the risk that there are just by chance sequences somewhere that allow that. But considering you see them in both I am pretty sure that they are typical artifacts. They are also stronger in the cauliflower because the specific target is missing that would outdilute the primer for the PCR reaction.

Edited September 29, 2013 by CharonY

[Genecks]

Because my knowledge is rusty; because the load involved DNA. Thank you for chiming in.

Edited September 29, 2013 by Genecks

[Tridimity]

Hi there

I need some help with a PCR experiment I had to do for an assignment and I'm not sure if I completely understand whats happening in the agarose gel photo. I've added the gel photo and also the molecular weight marker used.

 

lanes (from left to right) 1=negative control, 2=chicken DNA, 3=cauliflower DNA, 4= Molecular weight marker

 

this experiment was to see if using the same primers produced from cauliflower beta-actin, and by adding them to chicken DNA if they would amplify the same sequence.

 

I would expect it not to amplify the same, and the gel photo shows the brightest chicken fragment higher than the cauliflower. but then there is also a fragment that is at the same height as the cauliflower but not nearly as bright.

 

I'm still trying to wrap my head around all this and not sure about what i'm actually seeing, if someone could help me on the right path that would be great!

 

Cheers

Ashlee

 

 

I'm trying to follow your logic.

 

It appears that you have developed a Southern Blot. You have four lanes. Each lane is discussed, as you have previously mentioned.

 

I'm stuck at this next part. Let me see if I understand.

 

Someone has added the DNA for Cauliflower beta-actin into the Chicken DNA. I am not sure how the methodology went about this.

 

Was this a cell that had DNA of chicken and cauliflower in it?

 

Was the second lane from an extract of a Chicken DNA, which then had the primers that are later translated and transcribed in cauliflower cells in order to make cauliflower beta-actin, separately loaded with the Chicken DNA, thus causing the second lane to have a mixture of Chicken DNA and cauliflower beta-actin primers?

 

Another thing not mentioned is the molecular weight of various things you expect to see.

 

If the cauliflower primers bound to the chicken DNA, then yes, you're going to see a more amplified spot higher up on the molecular marker spot. If when cutting the hybrid chicken/cauliflower DNA with Hae III, it will then cut a the ends with the chicken and cauliflower DNA, then yes, you're going to get a higher molecular weight.

 

I'm not sure if I'm seeing things right here, though. Perhaps another member can chime in.

 

 

Southern blot? Looks like a standard agarose gel to me - Ashlee please can you confirm?

 

I think the point of the experiment was to extract and purify chicken (Gallus gallus) versus cauliflower (Brassica oleracea) genomic DNA and to attempt to PCR-amplify the gene encoding beta-actin, for both chicken and cauliflower samples, using primers that had been designed and were known to amplify beta-actin in cauliflower. A PCR-product of similar size resulting from the rxns using samples from the two different species would be indicative of a high degree of homology between the sequences.

 

From the looks of your gel, something has been amplified of size ~200bp in lane 3 (unless the band is due to primer-dimers). Lane 2 contains multiple bands - this may be due to non-specific binding of the primers - you could attempt to rectify this by increasing the annealing temperature. One of the bands in lane 2 (though faint) is of similar/equal size to that in lane 3.

 

I am unable to find the DNA sequence for Brassica oleracea beta-actin; the DNA sequence for Gallus gallus beta-actin (5,046 bp) is available from NCBI:

 

http://www.ncbi.nlm.nih.gov/nuccore/X00182.1

 

It would help if we knew the sequence of your primers and the conditions of your PCR rxns.

 

For future reference: if, when putting the gel into the gel imager, care is taken to avoid pockets of air between the gel and the surface of the imager, it will help to prevent air bubbles like the one in the middle of this gel. It may be worth playing around with the exposure to give greater contrast between the bands.

 

Hope this helps

[CharonY]

I realized that I misread the post. For some reasons I assumed that that the primer were derived from chicken rather than cauliflower, which pretty much invalidates my post. In that case it really depends on the primer how to interpret what you see. I.e. what area is targeted as they are highly conserved regions in beta-actin genes which could yield identical results across species. I.e. using the correct primers you could either amplify a region that is conserved across species or specific for only closely related ones.

 

For that you would have to know what fragment size to expect. Considering that you ran a high-percentage gel (as seen from the marker) I now suspect that the smaller fragment is indeed the expected one. As there is a bit smear up to the top, is it possible that you either contaminated or had a large amount of DNA in your PCR reaction? The alternative is, as Tridimity mentioned unspecific amplificates (of greater length that is).