Inhibitory component of a PCR assay - (Mar/05/2007 )
Hi
I have isolated DNA from cotton leaf samples. There are something which remains in well during agarose electrophoresis. and I also observed that this something is inhibitory for my PCR assay. Is it salt/ polysaccharides/ polyphenols........? It looks like an intense DNA band. So what is it exactly which also get stained with ethidium bromide....? How it could be removed ?.
Any suggestions are most welcome.
thanks !
Could you post a photo w/ pcr protocol, cycles and method of DNA purification???
i think the EtBr staining is due to the contamination holding back DNA within the well.
If I had to choose I would say it is polysaccharides/ polyphenols/charged polysaccaride.
You will have to improve your DNA extraction protocols. What is your current protocol. Please give details. I am sure the forumite here will be willing to suggest possible improvements.
As for the PCR, have you tried diluting your template DNA 1:10 to 1:50 dilution? Often you can dilute the contamination away and use PCR to amplify the very weak signal. Adding more Mg ion (50% inceases) can improve the signal (though the down side will be stronger secondary PCR bands)
DNA ISOLATION were carried out thru CTAB. Here I am attaching the photo of isolated DNA samples.
I don't know what exactly remains in the well, but surely it is inhibiting the PCR assay.
Thanks for kind reply
If I had to choose I would say it is polysaccharides/ polyphenols/charged polysaccaride.
You will have to improve your DNA extraction protocols. What is your current protocol. Please give details. I am sure the forumite here will be willing to suggest possible improvements.
As for the PCR, have you tried diluting your template DNA 1:10 to 1:50 dilution? Often you can dilute the contamination away and use PCR to amplify the very weak signal. Adding more Mg ion (50% inceases) can improve the signal (though the down side will be stronger secondary PCR bands)
Thanks for reply
After dilution, there was no amplification and even Mgcl2 at the coc of 1.5mM gives so many secondary bands.
ooo.. looks like you DNA is also mildly degraded.
CTAB.... umm that doesn't make a protocol. What is your protocol exactly? Do you use any thing to extact the phenolic compounds? What about the RNAse treatment? Write it all down. Because two CTAB protocol may not and probably will not be exactly the same.
And the devil is in the details.
CTAB.... umm that doesn't make a protocol. What is your protocol exactly? Do you use any thing to extact the phenolic compounds? What about the RNAse treatment? Write it all down. Because two CTAB protocol may not and probably will not be exactly the same.
And the devil is in the details.
Thank U Sir for reply
Here I have detailed the DNA extraction protocol from cotton leaves
1. Homogenize 25 mg of plant tissue in a mortar with 1.5 mL of extraction buffer. Transfer the mixture into a microcentrifuge tube.
2. Incubate the mixture at 55 °C for 30 min with frequent agitation, avoiding the suspension to settle. Cool down to room temperature.
3. Centrifuge at 16000 g for 10 min at room temperature. Transfer the supernatant to a new tube.
4. Add 1 volume of chloroform-isoamylalcohol (24:1) to the supernatant and vortex thoroughly.
5. Centrifuge at 16000 g for 10 min at room temperature. Transfer the aqueous (upper) phase to a new tube. Repeat the chloroform-isoamylalcohol extraction two times.
6. Transfer the supernatant to a new tube, add 0.45 volume of isopropanol and mix by inversion. Incubate at RT for 1 hour.
7. Centrifuge at 700 g for 10 min at room temperature. Discard the supernatant.
8. Wash the pellet by adding 1 mL of wash buffer and vortex. Centrifuge at 900 g for 10 min at room temperature.
9. Discard the supernatant and air dry the pellet at room temperature, but do not over dry. Dissolve the pellet in 25 µL of TE buffer.
10. Store the DNA solution at 4 °C for short-term or at –20 °C for long-term storage. If needed, treat the DNA solution with RNase before use.
11. Read absorption spectra at 230, 260 & 280nm. The ratio 260/280 should between 1.8-2.0 and 260/230 should be more than 2.0
Extraction buffer: 100 mM Tris-HCl (pH 8), 2.0 M NaCl, 20 mM EDTA (pH 8), 2 % (w/v)
CTAB, 1 %(w/v) PVP (PVP K10, MW 10.000), 0.5 % (w/v) of activated charcoal
Wash solution: 15 mM ammonium acetate in 75 % (v/v) ethanol
TE buffer: 10 mM Tris-HCl (pH 8), 1 mM EDTA (pH 8).
=> No RNase treatment was done.
=> Absorption spectra was also fine. But what exactly remains in the well is still a question. And somehow as I think this is the only inhibitor of PCR assay.
=> Sometimes after using HotStart Taq, there were some amplification but that was not consistant.
=> My PCR assay is fine as I have tested it thru positive control using same gene from bacterial DNA.
So this is total whatever I am using.
please guide me.
thanking in anticipation
What I see is that maybe in the final steps the DNA is not as clean as it should be and also you could improve the quantity if you change the volume of alcohol. For the precipitation step I'll suggest that use 2.5 vol of cold alcohol (.45 is to few volume, need a higher volume to make sure that you are diluting any trace of chemicals plus make the DNA precipitate). The alcohol could be absolute ethanol or isopropanol (I had use both and no difference) but the key is that is better cold (keep at -20c if possible). Other important thing is incubate at very low temp for a longer period of time (I had better results leaving overnight at -20C). If want a higher quantity also can add 0.5 vol of NaAc (3M) to the alcohol/sample. After incubation centrifuge at high speed for 10-15 min 9you must see a good pellet), discard supernatant and add 1mL 70% ethanol to wash pellet (tap the tube to make sure the pellet is dislodge(?)(well i mean not adhere to walls), centrifuge again for 5 min, repeat the step, discard supernatant and make sure no trace of alcohol (alcohol inhibits the PCR reaction). I suggest that use speed vacumm dryer at low temp if available if not could use air dry of an oven (at low temp!!! 40-50C is ok). To avoid the DNA degradation add proteinase K or an available enzyme to degrate dnase and other proteins or the proteins will do their work before you can eliminate them with the organic extraction. Hope this help!!!!
Aside from the above, this protocol is also missing, the RNAse step, the phenol/chloroform step
1. Homogenize 25 mg of plant tissue in a mortar with 1.5 mL of extraction buffer. Transfer the mixture into a microcentrifuge tube.
2. Incubate the mixture at 55 °C for 30 min with frequent agitation, avoiding the suspension to settle. Cool down to room temperature.
(I believe it would be better if you used 68°C.)
3. Centrifuge at 16000 g for 10 min at room temperature. Transfer the supernatant to a new tube.
4. Add 1 volume of chloroform-isoamylalcohol (24:1) to the supernatant and vortex thoroughly.
5. Centrifuge at 16000 g for 10 min at room temperature. Transfer the aqueous (upper) phase to a new tube. Repeat the chloroform-isoamylalcohol extraction two times.
RNAse step
Phenol chloroform extraction. Phenol/Chloroform/isoamylalchohol : 25:24:1, buffered at pH8. Repeat until interphase is clear.
6. Transfer the supernatant to a new tube, add 0.45 volume of isopropanol and mix by inversion. Incubate at RT for 1 hour.
I think it would be better if you used 3x volume 100% ethanol and 1:10 volume of 3M sodium acetate and incubate at -20 Celsius for 20min. Or better yet use -80 100% ethanol.
7. Centrifuge at 700 g for 10 min at room temperature. Discard the supernatant.
(Why don't you use a higher g force? and do the centrifuge at 4 celsius? All this work at room temperature will give the nuclease extra speed in degrading your DNA.)
8. Wash the pellet by adding 1 mL of wash buffer and vortex. Centrifuge at 900 g for 10 min at room temperature.
9. Discard the supernatant and air dry the pellet at room temperature, but do not over dry. Dissolve the pellet in 25 µL of TE buffer.
10. Store the DNA solution at 4 °C for short-term or at –20 °C for long-term storage. If needed, treat the DNA solution with RNase before use.
11. Read absorption spectra at 230, 260 & 280nm. The ratio 260/280 should between 1.8-2.0 and 260/230 should be more than 2.0
Had you think to use another protocol without organic extraction??There are some kits that don't use phenol/chloroform/isoamyl, they are less hazardous for your health and more enviroment friendly. For several years I used organic extraction, but after discovering those kits I prefer them.