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Restriction enzyme - PCR sheep blood gDNA


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#1 agorganic

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Posted 26 November 2012 - 05:18 AM

G'day,

I'm working on my research project which formed part of a thesis and need to spend a few days of the next week or so to finish up the last phase of the laboratory work by adding the restriction enzymes to determine whether or not a specific gene is present in my samples.

I have 70 gDNA samples (from ewes each bearing a single foetus), although I made an error in pippetting the gDNA in about half (33) the PCR plate wells and rather than adding 5ul, I ended up adding 50 microlitres of gDNA in with the PCR master mix 1 (made up of redtaq polymerase/primer/buffer/H20 mix, which 45ul is added into each well of the PCR plate). Therefore there is a total volume of 95ul in each well for half (33) of the samples and I've used up a significant amount of the Redtaq in stock, so can't go back and repeat these. The PCR wells with the correct volume have 5ul of gDNA along with 45ul of the master mix 1.

Yet to be made is Master mix 2 for my restriction enzymes (AVAII), which the documentation is attached.

A lab tech who has been assisting suggested making the master mix into a new PCR plate using the following method:

5ul of PCR master mix 1 (gDNA) into each well and resuspend
2ul of restriction enzyme
3ul of buffer
15ul of H20

My project supervisor suggested the restriction enzyme would be too much, if I can conserve it without influencing my downstream analysis, then that would be great. Except I'm not overly familiar with this and time for error is closing fast. If anyone has any other suggestions (calculations factoring in a couple of ghost samples for spares).

Depending on how long this takes me tomorrow morning, I might initially run 12 samples with the master mix 2 and put into the thermocycler, although my supervisor recommended to run the program for as long as I could. The thermocycler will sit at 12 degrees C when complete. Then I'll run twelve samples on the agarose gel, but I've got at least 34 samples out of 70 that I could test. 2 of the 70 were intended as dirty [whole blood] PCR and hadn't shown up in my gel electrophoresis, having wanted to use kangaroo (sex unkown) and a sheep from a different farm as controls.

I'll run a ladder, which is quite big, though something to compare with; along with a non-digested enzyme, gDNA, PCR (master mix 1 only) and then the digested enzymes master mix 2 samples.

If someone could provide feedback on what master mix to use instead of the one above, that is in line with the AVAII restriction enzyme procedure, then that would be great.

Jesse

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#2 agorganic

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Posted 26 November 2012 - 11:42 AM

Also I need to add the 6x loading dye. The gel has got the ethidium bromide in it.

#3 Trof

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Posted 26 November 2012 - 11:42 AM

Um, are you going to restrict your PCR product?

Did you see your products on gel? That's the first thing to do before restriction, if you crewed the PCR setup, those samples with 10x more DNA may not amplify at all and it would be a waste to try to restrict them. You still only use 5 ul of 50ul reaction anyway, so you can put 10 ul on gel.

The amount of restriction enzyme needed is dependent on the amount of DNA used. But usually for fine cutter as AvaII, 1 ul ul will be enough for some 400 ng of DNA. But you can't put too little DNA in restriction reaction, because you wouldn't be able to see it on gel. How big is the product and how many times will AvaII cut it (what size of fragments).
If you have many same samples, I would try to find ideal amount of enzyme needed, (maybe even 0.5 ul will suffice) on one of the samples, and then use this amount for the others, but you need to use equal amount of DNA. Gel picture of products can help you estimate the product concentration.

As your "mix" for restriction. AvaII only needs buffer 4, template and temperature. I don't see reason why to try substitute any of these with something else.
First your calculation is not right, buffers are used as 10x diluted, but for 25 ul reaction you have 3 ul of buffer, that would be for 30 ul reaction, either increase the amount of water to have 30 ul overal or decrease the buffer (and recalculate water accordingly).
Second thing is, if you going to pipet in this order, it's wrong. Enzyme goes as last and definitely to the enviroment with buffer already diluted to proper concentration.
AvaII can cut for longer time, but not recomended longer than 8 hours. But reaction won't be stopped in 12 degrees, so either set reaction in the morning of just don't cut that long, If you don't have excess DNA, three hours should be more than enough. But the more time you use, the less enzyme you need (if the enzyme is stable for such long time).

Look up FAQs for AvaII on NEB pages, that can tell you more about unit requirement over extended digestions.

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#4 leelee

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Posted 26 November 2012 - 07:23 PM

1. just in case you aren't aware- the concentrations for all of your PCR reagents will be out in the wells where you have added 50ul of template instead of 5ul. Assuming that your template is not very concentrated, you might be ok, but a 10 fold increase in template (and possible PCR inhibitors) may cause your reaction to fail, especially as your PCR buffer won't be at the right concentration

2. like Trof has said, are you going to run some of your PCR product on a gel before digesting? It would be a massive waste of time and reagents to try and digest failed reactions, especially as you have so many

3. 1ul of restriction enzyme will most likely be enough (and yes, fix up the volume errors that Trof spotted)

4. when you say you are adding "PCR master mix 1 (gDNA)" to your digest, you do mean after PCR right? And what do you mean by "program" for your digest? I'm confused, are you just using the thermocycler because you can set the temperature accurately and can fit your plate?

#5 agorganic

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Posted 02 December 2012 - 11:51 PM

Hi fella's,

Thanks.

I ended up setting the thermocycler to finish @ 4 degrees C (forever). I set the electrophoresis for 70 volts over 30 minutes, the amps were set at 400, although the BIO-RAD powerpac seems to alter the amps depending on the volts entered when ran.

I was rightly disappointed when looking under the UV, it looks like there is a problem shown here, where the ladder is on the left and then the 5th well is the PCR'd gDNA sample with no restriction enzyme which was used as a control against the same sample which was restriction enzyme digested in thermocycler. Therefore all the other wells are not displaying anything....

The agarose gel I made had ethidium bromide in it. Except for the ladder, all samples placed into the well had the x6 loading dye, even the control.

My research supervisor suggested digesting with more DNA. I'll add some more useful information associated with this once I've had more of a chance to go through the finer details, as I'm now working in a casual position with a grain storage company over the southern hemisphere harvest.

#6 Trof

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Posted 03 December 2012 - 02:40 AM

Your specification of all you do is very unclear. Can you please write clearly what exactly is the whole protocol? You should check products on gel prior the digestion.
Then, the gel image is bad, it's unfocused and from the little I can tell from it, not even the ladder is distinguished correctly. What are the sizes of the ladder bands anyway? Your sample is way bellow even the first band, which suggest it's maybe not a product at all, but a dimer (but hard to say, not knowing what ladder you used, but I hope it's some that has relevant sizes for PCR product, at least).

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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