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I am new to sequencing LSU, and I find I have multiple bands on my PCRs (using LR5 and LROR with a two-stage PCR: 5 cycles annealing 61.5deg and 30 cylces at 59). Some come out with single lovely and sequencable bands of about 1000bp; some have two or three bands with 1300 or closer to 1800bp. The longest band is invariably nice and bright. I am expecting to find group I intron insertions to explain these extra bands (will be sequencing them shortly).
I would like to avoid cloning/gel-extracting and would like to hear others' experiences/ideas about this.
Thanks in advance
Rebecca Yahr, Royal Botanic Garden Edinburgh
If you have multiple bands with high concentration of DNA, it would be good at the beginning of your study to run a electrophoresis gel, cut every band and purify separately (even though that you comment that you want to avoid to do this). Thus, you will have a better idea about if you really have introns or not, also if the different bands are due to contaminations.
My experience with different groups of fungi is that sometimes some copies of the LSU have introns and other does not have them, even though they belong to the same isolate.
We have this problem from time to time and it's been due to intron presence of variable sizes. Usually, the introns are adjacent to the LR5 primer. To avoid excising PCR bands or cloning, you could try sequencing from the left (5' end) only using LR0R, then the intermediate primer LR3R or complements of LR16 or LR21 to try to sequence through the intron insertion area without encountering heterozygosity.
Thanks Karen. I will try some other primers for sequencing.
Could you please tell us what species/genera you are working on?
The optimal method of amplifying loci via PCR can vary per genera, also something like as your PCR mix can matter.
If you can provide us with some more info, you stand a better chance of us being able to help you.
I'm working on Usnea in the Lecanoromycetes. I have had some advice about PCR protocols, but even with the much higher annealing temperature I'm using, I'm still getting the multiple bands for some samples. Sequencing of these bands is one of my next steps to ensure they are not contaminants, but I would like to ramp up my throughput and try to avoid them in the first place.
If you really do not want to clone, there are several alternative methods to optimize your PCR and reduce your bands.
1) Use less DNA template in your PCR reaction, adding to much DNA could be messing up your PCR.
2) Use DMSO in your PCR reaction to inhibit secondary structures in the DNA template, this might give your template the boost it needs to amplify more specific.
3) Use touchdown PCR, starting from your optimal TM +10 degrees Celsius and go down 0.5 derees per cycle to your optimal TM (boost amplification of your most specific template) and than add 35 normal PCR cycles.
4) And if that does not work, just switch primer. I never had any problems with LR5 myself, but an unfamilliar with LROR. There are PLENTY of alternative LSU primers out there so changing LROR for an alternative primer will not be a problem. I personally like the combinations LSU1fd/LR5 or VG9/LR5 for Dothideomycetes.
I have experienced same band size with multiple amplicons, except cloning, I have figured out a easier method to solve this, keep everything but except doing 1:10 dilution of the DNA template, do a 1:2 and so on, ignore the beginning dilutions and start doing PCR after at least 512 or 1024x dilution, it works great for me without any effort on picking clones :)
Thanks Isaac - An interesting idea and not the first time I've heard it. I will try it with a few of the recalcitrant ones!
Working on Usnea, I bet you have multiple bands because of lichen endophytes. I always had that problem with ribosomal genes of lichens in the AFTOL project. I am afraid that cloning will be the only solution...
All the best,
I also tried to avoid contaminant amplification. The major problem is that lichenized fungi have often many more introns than the non-lichenized one and the PCR is biased toward the shorter fragment. With much more template DNA of the lichenized fungus, you will still amplify the contaminants.
Hi Val -
Nice to hear from you! Yes, I recall lots of cloning in V Reeb's lichen LSU life and I have no doubt there are lots of goodies living inside as well. THere are some interesting ideas floating around, so I will see where I can get with them. I've already ordered a big gel-extraction kit, so I'll at least know what I'm facing.
Nice to hear from you too...! I tried to modify my answer but apparently it did not work. I wanted to precise that even with dilutions or modifying the PCR program you will not avoid lichen contaminants. This problem is typical for ribosomal genes. As I mentioned, you have much more template of the lichenized fungus and for single copy genes that are the same size for contaminants and lichenized fungus, I almost never had this problem. Do you really need to recover LSU ?