I want to use DNA for RAPD PCR, i have only ever used boilates for PCR, but would it be better to extract the DNA using a kit?
Hello Jade. In my experience you can use simple crude lysates for PCR prepared using 0.05M NAOH by heating at 95C for 15 min followed by partial neutralisation with 1M tris pH 7.5. I routinely perform this method of extraction for PCR genotyping which has the advantage of being quick cheap and direct. However the DNA is not purified and thus contaminated with salts and also potentially DNAses. Thus we use this method for short term DNA preps. For longer term storage properly purified DNA using columns is advantageous by virtue of removing salts and protein including DNAses. It also by virtue of producing better quality DNA tends to give cleaner and more efficient amplification and thus is particularly good when target sequence and/or primers are less than ideal. However it is less direct slower and more expensive. For most simple diagnostic PCRs crude lysates will therefore suffice but in the event of poor and/or non specific amplification supplementary column clean up is an optional troubleshooting tool
Hello Jade. In my experience you can use simple crude lysates for PCR prepared using 0.05M NAOH by heating at 95C for 15 min followed by partial neutralisation with 1M tris pH 7.5. I routinely perform this method of extraction for PCR genotyping which has the advantage of being quick cheap and direct. However the DNA is not purified and thus contaminated with salts and also potentially DNAses. Thus we use this method for short term DNA preps. For longer term storage properly purified DNA using columns is advantageous by virtue of removing salts and protein including DNAses. It also by virtue of producing better quality DNA tends to give cleaner and more efficient amplification and thus is particularly good when target sequence and/or primers are less than ideal. However it is less direct slower and more expensive. For most simple diagnostic PCRs crude lysates will therefore suffice but in the event of poor and/or non specific amplification supplementary column clean up is an optional troubleshooting tool
Just wanted to add that it depends on your bacteria - boiling gram negative (few colonies in water, 10min at 95°C, centrifuge) will definitely give you nice enough DNA for PCR.
Gram prositive are a bit harder, so would recommend a kit there.
Hi Laurence,
Can this direct method be applied for DNA extraction from gram positive bacteria? I am practising boiling method using saline for gram negative bacteria DNA extraction. These extraction are for PCR analysis and for short period storages ( maximum a week).
Which method is preferable for gram positive bacteria?
Please suggest
Dear Lakshmi
I will let others who have more direct experience with gram positive and negative bacteria comment on different approaches
Hi Jade ,
I think that the boil is a good and cheaper method for recovery bacterial DNA, but the DNA obtained is "dirty" not suitable for a RAPD PCR. I think that the boil extraction not perform every time in the same manner, and the RAPD PCR is "susceptible" method with some criticity, I don't would like to ever complicate a method already critic!!! so for your tranquillity and for good reproducibility I think that the right choise be the use of an extraction kit!!
good work
Hi Jade,
We've been doing boiling lysis this to screen a large quantity of bacterial isolates. If it was more than enough to produce good quality 16S rRNA PCR products for sequencing, it was really crappy for RAPD-PCR. Same for other "random-ish" type of PCR such as ERIC, BOX, etc.
The same RAPD primers worked consistently though when DNA was extracted and purified, then quantified and used in the exact same amount per PCR reaction.
Nota: you can also have different RAPD patterns when running the PCR on two different thermocyclers...
some teams boil the samples, or use hot organic extraction. I'd advise to use the downstream purification step. Try eg the new PureLink Microbiome DNA purification kit - it has protocols for tough samples like soil, stool, etc. works really well for microbiome applications- bacteria fungi etc
I cannot add to the above: boiling for 5 min in dilute NaOH will result in a crude lysate that is usually if sufficient quality in my experience for genomic based PCR
I suppose the OP has now solved her two years old problem. Note also that she was explicitly asking for RAPD PCR
Good day everyone!
By boiling do we mean 100 degrees centigrade?
I am cureently working with cryptosporidium and cyclospora oocysts. normally they use repeated ( 8 to 12) freeze thaw cycles then prolonged incubation in proteinase K (3 to 18 hours in 50 to 60 degress centigrade) just to rupture the oocyst and release the coccidian DNA.
Do you think that straight boiling (100 degrees centrigrade would be enough to destroy an oocust and release DNA? then the eukaryotic DNA released would be denatured (separated already) so will it still be useful for simple PCR?
I hope you can share all your ideas.
thanks so much in advance.
Hi, I have not experienced with cryptosporidium and cyclospora oocysts, but I used the boiling with Cryptococcus and the results are good. After the boiling the DNA do not stay denatured but you can use the crude lysate for performed any PCR do you want !! So I think that you haven't nothing to do that try it !! let me know!
bye bye and good work!
ps. keep on mind that the time of boiling is important, sometime a long time at high temperature do not uesful to try with different time of incubation can improve the final results
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