Previously we used to use RNAse free water (DEPC water) for all genetic experiments that is RNA extraction, cDNA synthesis, semi-PCR or RT-PCR. Recently we use DEPC water only for RNA work. For cDNA and Primer dilution we use water (filtered). One of experienced researcher changed our protocol. I guess primers are susceptible with water (though DNA is stable)? What would be your suggestion in such matter?
I agree with both previous commentators: commercial Molecular grade water certified as RNAse and DNAse free is always your best option; What is more filter sterilising removes potential particulate matter (contamination) that could contribute to degradation; A viable alternative is to use filter sterilised and preferably autoclaved and then filter sterilised milli Q water which by definition can be inferred to be DNAse and RNAse free and thus should not degrade primers, cDNA etc
Therefore either type of water unless contaminated is suitable for primers RT synthesis etc. If problems are experienced don't change the type of water you use; Rather the aliquot
Routinely for both types of water I will change my aliquot regularly and in the case for example of molecular grade water, i.e. certified to be both RNase and DNase free I do not put tips or pipettes into the commercial stock bottle; Rather pour out regular small aliquots into molecular grade plastic ware for limited use. This minimises contamination with particulate contaminants which might carry RNases and DNAses
Where Milli Q (18.2MOhm) water is concerned failure to change the cartridges regularly can lead to alterations in pH which in turn can lead to breakdown of DNA species like cDNA and primers (and RNA) by hydrolysis. This hydrolytic breakdown of DNA & RNA is quite separate from enzymatic breakdown of DNA and RNA by contaminating DNAses and RNases respectively: The former is controlled by using commercial grade water of a certified pH or by Adding 10mM Tris pH 8.0 to your primer stock diluent (for example) and/or by keeping regular checks on the resistivity values of your Milli Q ion exchange cartridges; The latter by filter sterilising and/or autoclaving water to remove particulate contaminants that can absorb DNAses and RNAses
Incidentally autoclaving will destroy DNAses but NOT RNAses which require temp of > 180C for > 5 hours; Preferably in conjunction with DEPC. Regarding a prior comment made about DEPC, yes that is indeed correct if the DEPC itself is not removed by autoclaving but with autoclaving DEPC is readily and efficiently broken down into CO2 and water by autoclaving so should not be an issue
In summary:
1. In the case of primers make up 100uM stocks in either commercial grade water certified as RNAse/DNase free. Take aliquots of this water by DECANTING from the stock bottle rather than constantly re introducing tips and/or stripettes. This will minimise contamination of your stock solution
2. Better still for storage of stock primers for >/= I year dissolve in 10mM Tris pH 8.0 + 1mM EDTA pH 8.0 that has been autoclaved and filter sterilised. Primers are stable for prolonged periods (> 1year) with TE because they are not prone to slow insipid hydrolysis caused by shifts in pH
3. make working stocks of primers by diluting the above archived stocks 1/10 with Molecular grade water filter sterilised and preferable autoclaved. Keep these working stocks for no more than 1 month (at 4C) and then discard
4. Alternatively use autoclaved filter sterilised Milli Q water
5. For both types of water take regular small aliquots and discard on a regular basis
6. DO NOT dilute your working stocks of primer with TE (EDTA can interfere sometimes with down stream reactions like RT but is effectively diluted to levels with water in working stocks where this effect is not apparent)
7. Both types of water can be used for cDNA reactions etc as well as primer stocks
8. Regarding DEPC water make by adding 1ml of DEPC (< 3 months old; It hydrolyses in the fridge so try and keep in a desiccated container or seal the top of the bottle with parafilm) to 1 litre of ddH2O, stir in a fume hood over night; Shake the following morning and place at 37C for 1 hour; then autoclave. With autoclaving it should be safe to use for standard molecular reactions (unless you precipitate DNA and RNA with Ammonium salts like ammonium acetate or ammonium chloride. Hence the above commentators comments) but in truth it is not now considered necessary if the above protocols are adhered to
Nuclease free double AMQ does good for Primers as well as cDNA. But try to avoid DEPC water which might interfere in down stream assays.
I agree with both previous commentators: commercial Molecular grade water certified as RNAse and DNAse free is always your best option; What is more filter sterilising removes potential particulate matter (contamination) that could contribute to degradation; A viable alternative is to use filter sterilised and preferably autoclaved and then filter sterilised milli Q water which by definition can be inferred to be DNAse and RNAse free and thus should not degrade primers, cDNA etc
Therefore either type of water unless contaminated is suitable for primers RT synthesis etc. If problems are experienced don't change the type of water you use; Rather the aliquot
Routinely for both types of water I will change my aliquot regularly and in the case for example of molecular grade water, i.e. certified to be both RNase and DNase free I do not put tips or pipettes into the commercial stock bottle; Rather pour out regular small aliquots into molecular grade plastic ware for limited use. This minimises contamination with particulate contaminants which might carry RNases and DNAses
Where Milli Q (18.2MOhm) water is concerned failure to change the cartridges regularly can lead to alterations in pH which in turn can lead to breakdown of DNA species like cDNA and primers (and RNA) by hydrolysis. This hydrolytic breakdown of DNA & RNA is quite separate from enzymatic breakdown of DNA and RNA by contaminating DNAses and RNases respectively: The former is controlled by using commercial grade water of a certified pH or by Adding 10mM Tris pH 8.0 to your primer stock diluent (for example) and/or by keeping regular checks on the resistivity values of your Milli Q ion exchange cartridges; The latter by filter sterilising and/or autoclaving water to remove particulate contaminants that can absorb DNAses and RNAses
Incidentally autoclaving will destroy DNAses but NOT RNAses which require temp of > 180C for > 5 hours; Preferably in conjunction with DEPC. Regarding a prior comment made about DEPC, yes that is indeed correct if the DEPC itself is not removed by autoclaving but with autoclaving DEPC is readily and efficiently broken down into CO2 and water by autoclaving so should not be an issue
In summary:
1. In the case of primers make up 100uM stocks in either commercial grade water certified as RNAse/DNase free. Take aliquots of this water by DECANTING from the stock bottle rather than constantly re introducing tips and/or stripettes. This will minimise contamination of your stock solution
2. Better still for storage of stock primers for >/= I year dissolve in 10mM Tris pH 8.0 + 1mM EDTA pH 8.0 that has been autoclaved and filter sterilised. Primers are stable for prolonged periods (> 1year) with TE because they are not prone to slow insipid hydrolysis caused by shifts in pH
3. make working stocks of primers by diluting the above archived stocks 1/10 with Molecular grade water filter sterilised and preferable autoclaved. Keep these working stocks for no more than 1 month (at 4C) and then discard
4. Alternatively use autoclaved filter sterilised Milli Q water
5. For both types of water take regular small aliquots and discard on a regular basis
6. DO NOT dilute your working stocks of primer with TE (EDTA can interfere sometimes with down stream reactions like RT but is effectively diluted to levels with water in working stocks where this effect is not apparent)
7. Both types of water can be used for cDNA reactions etc as well as primer stocks
8. Regarding DEPC water make by adding 1ml of DEPC (< 3 months old; It hydrolyses in the fridge so try and keep in a desiccated container or seal the top of the bottle with parafilm) to 1 litre of ddH2O, stir in a fume hood over night; Shake the following morning and place at 37C for 1 hour; then autoclave. With autoclaving it should be safe to use for standard molecular reactions (unless you precipitate DNA and RNA with Ammonium salts like ammonium acetate or ammonium chloride. Hence the above commentators comments) but in truth it is not now considered necessary if the above protocols are adhered to
Hi Gaire,
For RNA work, using DEPC treated solutions (water or other solutions) is highly recommended. DEPC by covalent modification inactivates RNases. But always keep in mind that residual DEPC if remains in the solution can affect many things like modification of purine residues in RNA etc. So eliminate residual DEPC by autoclaving or heating to 100 degree celcius for at least 15-20 minutes.
For cDNA and primer dilution, autoclaved MQ water is the best choice in my opinion.
All the best!
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