7 replies to this topic

[sligeach]

I am currently trying to genotype some transgenic mice. All of my previous experience with pcr has been working with RNA therefore i thought that when working with DNA it would be a breeze however i have been proved wrong. I got tail clipings from my mice and then because i have moved to a new lab i had not everything i wanted to do the DNA extractions myself so i sent the samples of to a department in the unviersity to do it. The protocol which they used for the isolation of DNA i have attached.

The people i sent my samples to for DNA isolation also measured the DNA concentration for me via a nanodrop.

To my frustration there is massive variation in the "quality" or suitability of the extracted DNA for PCR as assessed via the use of a pair of primers which i use as a positive control obtained from the jackson lab which they use for their PCR experiments on genotyping mice. For the quality checks i used 250ng of DNA with a primer concentration of 0.5um and preformed 40 cycles. In some samples i get a very strong band while in others i get either very faint bands or no bands at all. The PCR kit i use is the roche fast start pcr master catalogue number 04710444001.

What i am intersted to hear about is do people have any comments on the DNA extraction protocol which was used and do most of ye who do genotyping experiments use commercial kits to purify your DNA which you will use for PCR experiments if so are there any ones you reccomend.

Thirdly have any of ye tested different PCR kits with the same DNA template and found any differences between manufacturers in their ability to produce a succesful PCR over an alternative supplier?

Attached Files

•  DNA EXTRACTION PROTOCOL.docx   5.26K   90 downloads

[bob1]

The protocol is fine, it appears to be a standard DNA extraction with a chloroform step.  I have used similar protocols quite a bit for genotyping and never had any problems with the DNA not amplifying.  I have even done protocols which skip the chloroform step and just do a salt precipitation of the protein then ethanol/IPA pptn of the DNA with no problems downstream.

Try diluting the DNA 1:10 or 1:50 and see if this works.  This will often dilute out inhibitors that are stopping the amplification.  Poor technique when doing the phase separation will carry over chloroform into the later steps, which could be a problem.

Just to check: are your negative controls negative?  Also, have you tried repeating the samples with poor amplification and got the same result?

[sligeach]

thanks for your reply bob1

Yes my negative controls are negative

I have tried multiple different primer sets 4-5 and i keep getting the same pattern in that for the samples which give a good signal for my positive control primers i get expected bands with the other sets of primers while for the samples which give low or no signal with the positive control primers i typically dont get any signal with the other primer sets. It was the latter finding which made me realise it must have something to do with the actual DNA template and this was confirmed with the positive control primers.

i was thinking of doing a dilution and seeing what would happen.

For your genotyping experiments bob1 do you have a preference for a particular pcr kit or DNA extraction method and also what concentration of DNA and primers do you typcially use. I had a 25ul pcr reaction with 250ng of DNA and 0.5um primers.

[bob1]

I usually did (I don't work with the mice anymore) a basic proteinase K digest overnight and then protein ppt followed by DNA ppt, so no kit based methods.  However, the genotyping system was already up and running well by the time I got there.

I can't remember the exact PCR conditions, but the ones you are using look like normal genomic DNA conditions.  I presume you have optimised the reactions for the other primers?

[sligeach]

yes from my work with RNA and qPCR i learned very early the importance of always running temp gradients to optimise the primer sets and i also test out different primer concentrations of 0.5 1 and 2um as from my experience some primer sets need a higher concentration in order to give me consistent and satisfactory detection at least for lowly expressed mRNA transcripts.

For genotyping experiments im beggining to think it may be worth the extra expense of buying a commerical DNA extraction kit in order to ensure or at least improve the likelihood of me getting consistently useful DNA for PCR experiments as the amount of time i have spent trying to get the current DNA samples working is not cost effective as ive had to use signficantly more of my time and PCR kit than i had planned.

If anybody has experience of using a commercial DNA extraction kit and found it to consistently give them good quality/pure DNA from mouse tail extractions for subsequent PCR then i would be very interested to hear about it.

[Trof]

We use the Qiagen kit for mouse-tail DNA.
However even then, big problems with inhibition. We genotype by HRM, we need very little concentration (around 7 ng/ul) so we dilute, and everything works fine.
But even when we got mouse-tail DNA from other lab that uses phenol:chlorophorm, they had big issues with PCRing it.
I diluted it to 10 ng/ul and used more cycles to compensate this and I had no problems with it.

[sligeach]

thanks for all the suggestions. I thought i would just let people know who may be having similar problems to what i had - that i decreased the DNA concentration to 10ng per 25ul total volumer pcr reaction and most of my PCR reactions worked reasonably well. there are still a few samples that are not working very well so am still trying to optimise these.

[Myworkismyplay]

Doing a simple alkaline digest of tail snips or ear punches provides plenty of gDNA and without all the inhibitory ingredients from PK digestion, and takes much less time, as well as saves tons of $$$.

Add 0.5mm tail or 1-2mm ear punch to 30-40 uL 10mM NaOH. Heat to 95C for about 30-45 minutes (shaking at 500rpm, or vortex every 5-10 minutes). Alternatively I've done 60C for 20 min before the 30 min 95C incubation to help in the solubilization of the DNA (not getting caught up in the insoluble proteins denaturing within the sample becoming insoluble). Vortex briefly to ensure sample tissue is sufficiently broken up. After the heat/mixing step, spin down & cool on ice. Add equal volume (30-40 uL) 10mM Tris, pH 7.5 to neutralize the pH (still leaving basic, for best solubility of DNA). Keep cold for subsequent protocols.

For PCR just use 0.5-2ul per 20ul reaction. You don't need to centrifuge the tail digest to remove impurities & pellet crap, just don't pipet up the blob for PCR. if it really bothers you you can centrifuge at 5-10 min at 10,000rpm (cold is better) & transfer the supernatantl to a new tube.

If your PCR reactions are optimized and primers work well, there shod be any issues for this to work for you.

We clip tails, digest, do PCR, and run our gels all in one day. It saves a ton of time and money. There's no need to purchase expensive DNA extraction kits/reagents for colony genotyping.

For those who just want purer DNA for whatever reason, throw in an ethanol precipitation/wash step, dry, & resuspend in TE.

Good luck!

Edited by Diana Albarado, 26 December 2012 - 08:45 PM.