no PCR product! any advice? - (Nov/26/2007 )

i have been trying to get some amplification of yeast ribosomal DNA. the source of the yeast is from beetle guts that have been smeared onto Whatman FTA cards.

i've tried different recipes and cycling parameters to no avail, although I am able to get some amplification from positive controls which consist of purified Yeast DNA that were also applied to FTA cards.

this is my latest recipe:

10 ul 5X Buffer
6 ul 25 mM MgCl2
4 ul 2.5 mM dNTP
2 ul each of 10 uM forward and reverse primers (received in JUNE 2004)
0.25 ul of 5 unit Taq polymerase
25.75 ul of UV sterilized water

the DNA is embedded in the FTA cards so i just use a 2mm cut out and place that into the reaction tube. The cut out is processed according to the Whatman's protocol using a purification reagent and rinses with TE Buffer.

the forward primer is AGTACCCGCTGAACTTAAG and, according to the manfucturer, the melting temperature is 58 degrees celsius
the reverse primer is TCCTGAGGGAAACTTCG but i don't remember the melting temperature of this one.

the cycling parameters i found in an article in which the researchers used these yeast primers and are as follows:

94 C x 3 min

94 C x 1min
55 C x 1min
72 C x 2min
for 5 cycles with -1 degree drop for each cycle until reaching 50 C

94 C x 1min
50 C x 1min
72 C x 1min
for 30 cycles

72 C x 10 min


apologies for all the detailed information but i've gotten so many different explanations from many different people. if any one can provide any advice or thoughts on why i haven't seen any bands in my gel runs, I would appreciate it very much. i've gotten bands for a DNA ladder which show up beautifully, and weak bands for the positive control, but absolutely no bands for the samples that I want to amplify.

[quote name='smu2' date='Nov 26 2007, 04:55 PM' post='118098']
[quote name='antareez' post='118096' date='Nov 26 2007, 01:25 PM']

the beetle guts are smeared on the card and the yeasts should be in the beetle guts, if there are any, because they live symbiotically within the beetle guts. the Whatman protocol involves rinsing the cutout with a purification reagent and TE buffer that lyses cells and clears any cellular debris leaving all sorts of DNA bound to the fibers of the card. then one uses the cutout in a PCR reaction and amplifies the DNA of interest by using specific primers. in this case I am using universal primers used for amplifying yeast small subunit ribosomal DNA

Yeast cells are pretty tough. I would not trust the reagents in FTA cards to lyse yeast effectively. Something harsher, starting with zymolase treatment and phenol/chloroform extraction followed by ethanol precipitation would be the first thing to try, until you got results. Then you can try backing off in complexity as long as things still work. You could also think about a bead-beater approach to lysis.

oh, i should add that when i say all sorts of DNA are bound to the card I mean that any DNA present in the beetle guts will be bound to the card so that would include beetle DNA, yeast DNA and bacterial DNA. the cards are impregnated with chemicals that bind DNA to the fibers and protect it from UV, contamination, nucleases, etc.

as for the positive controls, actual yeast were cultured and purified to extract the DNA. the supernatant from the purified yeast was applied to the card so that in this case only Yeast DNA should be bound to the fibers of the card unless there was some contamination. but that wouldn't matter because the primers are designed to only amplify yeast DNA.

i thought that perhaps i'm able to get bands in the positive controls because there is a large amount of yeast DNA present from yeast cultures whereas from the beetle guts I would have a very small amount.

so, would it be correct to say that there is amplification of beetle gut yeast DNA but that the starting amount is so small that it does not appear in gel electrophoresis?? i wonder about that because a fellow student says that he often gets no bands when running a gel on his PCR product but he still has enough DNA present that he can send it away for sequencing.

I am unfamiliar with this method, however I was wondering have you tried a test run to see if you can amplify yeast DNA from yeast cells that you have spotted on a card. Yeast cells take quite a beating before they lyse, I am just concern that the lysis protocol was not effective enough. I am also slightly worried about the card, I assume that you are soaking a piece of watman paper in the PCR mix. Might there be PCR inhibitor on the card itself.

no, i have not tried that. it's interesting that more than one person has suggested stronger lysis of the yeast cells. that is certainly something to look into because i find it hard to believe that the Whatman purification reagent would universally lyse and clear all types of cellular material. however, it is supposed to be useful for plant material also and i imagine that plant cells are also quite tough. though, i don't know if they are tougher than yeast cells.

the following is the link to the Whatman FTA cards and protocol information

http://www.whatman.com/products/?pageID=7.31.31

unfortunately, the purification reagent is proprietary so i couldn't find out its components.

The wash agent is described in one of the patents. I forget what is in it right off hand -- maybe ethanolamine, but if you need to know, it is there.

In our lab, we sometimes do PCR directly from yeast cells that we obtain in a yeast two hybrid assay. It's not extremely efficient, but generally what we do is resuspend a small colony in 2 ul of water, then microwave for about a minute, then add the PCR reagents directly to the microwaved tube. You have to be careful not to get too much yeast in this protocol because that can mess things up, but this may not be a problem for you if you really think that there's not much there to start with. What I'm thinking you could do to modify this protocol is put your paper into a little bit of their elution buffer, then microwave it as we do, and then do your PCR directly from this. It would be fairly quick to try, and if it doesn't work then you could try the other suggestions of applying harsher conditions to lyse open the yeast.

Hope this helps.

I found an article online where they describe microwaving yeast on FTA cards for one minute. so, today i tried that.

i'll keep everyone posted.