I am trying to do a genotyping PCR for cep-1 in the TJ1 strain (which has a deletion in cep-1), and N2 (which has the wild type copy of the gene). For some reason, the single worm lysis protocol is working fine for the TJ1 mutants, but I’m having trouble getting it to consistently work for the N2 strain. Today I ran a PCR with 20 lysed worms, and only 1/20 lysed worms showed a band on the gel. I ran a positive and negative control with the PCR, and both came out as expected. Anyone know what might be going on? I also check under the dissecting scope if the worms are in the buffer before running the lysis. The Proteinase K was received by the lab 1/19/10. Maybe it’s getting too old? But I don’t think that would explain why it’s working with one strain and not the other.
Solutions: Worm Lysis Buffer (WLB)
50 mM KCl
10 mM Tris pH 8.3
2.5 mM MgCl2
0.45% NP-40 (IGEPAL)
0.45% Tween-20
0.01% Gelatin
Proteinase K stock solution
20mg/ml proteinase K
Protocol:
Add 5uL of proteinase K to 95uL of WLB
Pipette 5uL of WLB + proteinase K to a PCR tube
Pick a worm to the PCR tube
Freeze tube in liquid nitrogen for 10 mins
Heat tube at 65C for 60 min for lysis reaction
Heat tube at 95C for 15 min to inactivate proteinase K
Add PCR mix directly to the digested heat inactivated prep and cycle as usual
Yes, I have been using adult (gravid) worms for the PCR, sorry I forgot to mention that. This is also the age that I am using for the TJ1 strain. Is there a way to make the lysis PCR more reliable for this age group? I think I’ve read somewhere that doing multiple freeze-thaws before the lysis reaction helps
The lysis conditions should be fine (I’d usually freeze in a -80 freezer for five or ten minutes, and lyse at 60 degrees not 65, but I assume those aren’t the issues). I had no trouble PCRing from gravid worms. In any case, given that you’re able to get PCR to work consistently with the deletion allele, the problem is unlikely to be your lysis conditions. I assume you’re using the lysate fairly promptly, as crude lysate generally doesn’t store well?
My first suspicions would be that there’s something wrong with the primer stock you use to detect the wild-type product, or - if you’re using the same primers to detect the wild-type and the deleted products - that your PCR is struggling to amplify the larger product from the wild type version of the gene. If the PCR is more chancy for the wild-type gene, this could compound with other problems (for example with template quality). In either case, I’d recommend ordering primers; replacements for the wild-type-specific primers in the first instance, or in the second instance you should try ordering primers that amplify from the wild-type but not from the deletion, and that give a product of similar size to the product you get from PCRs that detect the deleted gene.
For this last PCR I actually stored the worm lysates at -20C overnight. I added the PCR mix and ran the PCR the next morning. I also had this problem when I ran the PCR shortly after the lysis reaction.
There’s something to be said about the primers I’m using… I don’t know their sequence (so I can’t check for primer dimers, secondary structure, etc…), and when I run it on the genomic controls I often get lower bands than expected, and higher bands than expected… I’m using a high fidelity polymerase that is “suitable” for long amplifications of 7.5kb of genomic DNA, so the polymerase shouldn’t be a problem. My N2 band shows up at ~4kb, and the deletion mutant band shows up at ~2kb. Also, I don’t know if this hurts, but the primer solution I’m using has both the forward and the reverse primers in the same solution. The wild type and the mutant are both using the same primer set.
Just to add my two cents for what it’s worth. I have found it very difficult to consistently get more than 2 kb products by single-worm PCR. I think this is due to the nature of the lysis and extraction of the DNA being so quick and dirty. If you can detect your deletion band, you should be able to still genotype by PCR to identify your mutant correct? If you want to make sure you have the WT band, you could isolate genomic DNA from a large amount of worms using phenol/chloroform. Just some suggestions. Good luck.
I found that crude lysate (used within ~1 hour or so of the lysis) gave DNA of suitable quality for long-PCR using a polymerase mix (my favorite was the Clontech Advantage cDNA kit, from a small number I tried); I regularly got 8-10 kb products that way. Still, using a proofreading polymerase mix suitable for long-PCR is wasteful if you’re just genotyping strains. Taq is much, much cheaper - though it doesn’t do a good job of amplifying larger products from crude lysate.
I would recommend that you order one (forwards) primer outside the sequence removed by the deletion, a second (reverse) primer within the sequence removed by the deletion that when used together with the first primer will give a product of 4-800 bp from the wild type, and a third (reverse) primer outside the sequence removed by the deletion that when used together with the first primer will give a product of 4-800 bp from the deletion allele, but will give a larger product or will fail to amplify from the wild type (using Taq). It might be possible to do the PCR with all three primers, but I always did separate PCRs to detect the wild-type and mutant alleles. If you need to find good primer sequences, I’ve always found the primers generated by Primer3 to be reliable.
I am using Phusion polymerase for this PCR, anyone know if it is a good polymerase for the application?
I also want to be able to figure out if I have a heterozygote with my PCR. Using my current primers, there is a really heavy preferential amplification of the lower band since it’s half the size. Do you think that if I used three primers, with one primer being ~800bp inside the deletion, that this might give me two clear bands if the genotyped worm is a het?
Phusion is a great, fast, and highly processive polymerase, but it’s expensive and I wouldn’t use it just for genotyping unless you have an infinite supply. It can also be finicky with low Tm oligos. I’d order new ones for no other reason than to know exactly what they are and what they do. I’d also stick to lower size amplicons from worm lysates.
If you use a 98°C melting temp with Phusion, I think evaporation gets to be an issue even with the hot bonnet. You can boost your PCR volume to 50-100ul or just use another polymerase with a smaller product.
I use the methodology of Hillel, but I definitely use all 3 primers in a single reaction. The one inside the deletion is referred to as the ‘poison’ primer because it kills the larger WT band the outer primers would generate. If you use all 3 primers at the same time, make sure that the WT and deletion bands are at least 100bp different in size - ideally you’d have a 400-500bp and a 600-700 bp.
I have had success with Lucigen’s Econotag, but it definitely won’t work with anything over 1kb from crude lysates.
I agree, Phusion is a great enzyme but it’s quite costly if you’re only using it for genotyping.
I usually use a ‘homebrew’ Taq for genotyping, but if it’s a troublesome template I use Qiagen’s HotStarTaq and it works well.
Hi there, I use the set of protocol in my lab for SW PCR and I had never failed with it. I would just share here the reagents and also the methodology if you still need it. For genotyping purpose, phusion is really not required, Taq will do a good job. and about the primers, I think you should order your own new pair of primers so that you would know what is your product. If its a deletion mutant, what I do was I order primers that flank the deletion region in the mutant ie forward primer at ~200bp upstream of the deletion and reverse primer ~100bp downstream of the deletion. Good luck.
Reagent: WLB
10mM Tris HCL
50mM KCL
1.5mM MgCl2
(pH8.3)
plus 3% proteinase K(10mg/ml)
-This WLB can be prepared in bulk and stored in -20
PCR master mix
Primer A 5uM = 3uL
Primer B 5uM = 3uL
dNTP 1mM = 3uL
pcr Buffer = 3uL
Taq polymerase = 0.5uL (<1kb) or 2uL (>2kb)
Top up with ddH2O to 27uL
Reaction
add 3uL of WLB in a PCR tube
pick a worm (gravid should works well)
heat tube to 65Celsius for 120min
inactivate proteinase K by heating to 95Celsius for 15min
Add in PCR master mix
run PCR
Why do some protocols suggest lysing worms at 65 degrees for 2 or even 4 hours? Is it because of volume of worms to be lysed? And is there any negative effect if you lyse worms for too long?
I don’t even use “worm lysis buffer” I just do it in the PCR buffer that I will ultimatly use for the PCR reaction. I think you can try many different things and do this many different ways and it will still work.
I also just use my PCR buffer now (and add proteinase K to it) and not the recipe listed above. Also, I recommend freezing the worms at -80C, and not at -20C. The colder freezer breaks up the worms better for PCR.
If you decide on 3 primer PCR, you absolutely want to have a control where you mix null and wt DNA (because that’s equivalent to a het) to ensure that you can get amplification of both the null and wt band (i.e. one isn’t “poisoning” the other).
Also if you want to look for potential off target effects or possible primer dimers, check out: http://www.ncbi.nlm.nih.gov/tools/primer-blast/
coolest little tool since the knife first sliced bread (ha ha)
Good luck.
Alright, I designed new primers and they seem to work much much better than the old ones (the lysis is working for all strains now).
I am doing a PCR to determine whether the worms are homozygous for the wild type or mutant deletion allele, or if they are heterozygous. When the worms are a het, the PCR amplifies a lot more of the shorter allele, so when I run it on a gel the het shows a very faint wild type band and a much brighter deletion band. Anyone know any tricks to fix the preferential amplification?
I find it helps to make the PCR more reliable if the worms are washed to remove bacteria before lysing. I routinely pick animals (of any age) into PCR tubes containing ~150 µl of M9 containing 0.025% Tween-20 (Triton X-100 would also be fine). After the worms settle, aspirate most of the buffer, wash again with M9/Tween-20 if there’s a lot of bacteria, then wash with lysis buffer (basically PCR buffer with detergents; I omit the gelatin) containing 10 mM DTT. After the worms settle, aspirate most of the buffer and add additional lysis buffer containing Proteinase K, freeze at -80˚C, then carry out the lysis procedure.
Although many protocols call for lysing at 65˚C, 55˚C for an hour has worked much more reliably for me. Fermentas claims that that the enzyme becomes inactivated after 20 minutes at 65˚C. The optimal temperature for PK activity is not, as has widely been reported, 65˚C, but 37˚C. Higher temperature is used to inhibit nucleases and to partially denature the substrate proteins. The DTT facilitates digestion of the cuticle by reducing disulfide bonds.
I’m feeling a little dense here. I’m trying to do single worm PCR, but I… can’t understand how to make the worm lysis buffer (listed in the first post).
I’ve poked around the internet a bit, and everywhere lists the ingredients in mM and in %; I’m probably missing something, as I always thought that some kind of quantity or volume was useful in addition to the concentration in order to be able to mix up a desired solution.
If someone could explain or translate, I’d be most appreciative.
