Hello every one i am making dsRNA for that i need to extract DNA from PCR Product any body guide me how to get high concentration of DNA/ul because we need this DNA in as template if concentration is low further steps not proceed more thanks.
Dear Saad
to obtain high concentration from a PCR product the most efficient way is to ethanol precipitate. However, the modified method below takes account of the fact that small products, i.e. those < 100bp are often lost with standard precipitation so by adding MgCl2 to the precipitation mixture you selectively enrich for such smaller amplicons; In other words do not add unless you PCR products are 100bp or less. Also, the addition of glycogen increases efficiency of recovery, especially when you total yield is 1-5ug and also increases pellet visibility giving you confidence that it has not been lost during precipitation
Specifically:
1. Add 1/10 volume 3M Sodium acetate + 3 volumes of 100% molecular grade ethanol to your PCR product
2. IF PCR PRODUCT < 100bp add 1/10 vol 1mM MgCl2 = 0.1mM mgcl2 final concentration
3. PREFERABLY (although not absolutely essential) add 1ul of 1mg/ml to 20mg/ml (anything in that bracket acceptable) molecular grade glycogen: Adding glycogen as an inert carrier will increase recovery of precipitated product, particularly if 1-5ug order of magnitude and also render the precipitated pellet more visible, making washing with 70% ethanol easier. However, providing you can see the pellet which if > 1ug is possible this is not absolutely essential so you can if necessary precipitate with slat + ethanol alone
4. Take eluted PCR product plus salt + ethanol and Glycogen and incubate for 1 hour to over nigh @ -20C (if glycogen added you only need to incubate for 1 hour for efficient recovery but if convenient it wont harm to leave over night)
Spin @ 13,000rpm in an eppendorf for 15-20 min
5. Using a p200 remove most of the ethanol from the top of the aqueous layer down to the surface of the pellet. Now remove the remainder of the aqueous phase with a p20: If precipitated with glycogen the PCR pellet will be opaque white and very visible; If precipitated without glycogen the pellet will be smaller & white so hold eppendorf up to the light to identify pellet and remove aqueous phase as described
6. Now add 0.ml to 1 ml of room temp 70% ethanol
7. If glycogen added vortex the tube to detach pellet in order to more efficiently wash: The 70% ethanol wash id designed to desalt pellet; Vortexing to detach will increase desalting effectiveness and salt is more soluble in room temp ethanol (hence why you use RT ethanol and not ethanol placed at -20C)
8. If precipitated without glycogen, hold tube up to the light add ethanol gently down the sides, remove as described and add the same volume of 70% ethanol back
9. Now spin tube for 5 min at 13,000rpm in order to secure insoluble pellet to the side of the tube
10. Remove ethanol as before: This time however when you reach the level of the pellet remove the last few ul with a p10 tip
11. Now spin the tube for a further 1 min @ 13,000rpm to bring down the residual 70% ethanol up the sides of the tube. This normally amounts to a few ul
12. Remove this residual ethanol with a p10 tip
13. Now air dry the pellet for about 20 min @ room temp: When dessicated the pellet - unless contaminated with salt and protein - will turn clear, indicating it has dried
14. Now simply resuspend in a smaller volume of molecular grade water or TE (relative to starting eluted volume of PCR product) in order to make more concentrated
Good luck !!
HI,
I am not sure that you are using any kit or what kind of kit!!! If you extract DNA manually from gel i think using a very low concentration of agarose gel would be a very good idea. In case of kit u need a very good quality of Column (Midi-column) and Elution Buffer should be accurate in concentration (Fresh). ....
Thanks.
Dear Saad
to obtain high concentration from a PCR product the most efficient way is to ethanol precipitate. However, the modified method below takes account of the fact that small products, i.e. those < 100bp are often lost with standard precipitation so by adding MgCl2 to the precipitation mixture you selectively enrich for such smaller amplicons; In other words do not add unless you PCR products are 100bp or less. Also, the addition of glycogen increases efficiency of recovery, especially when you total yield is 1-5ug and also increases pellet visibility giving you confidence that it has not been lost during precipitation
Specifically:
1. Add 1/10 volume 3M Sodium acetate + 3 volumes of 100% molecular grade ethanol to your PCR product
2. IF PCR PRODUCT < 100bp add 1/10 vol 1mM MgCl2 = 0.1mM mgcl2 final concentration
3. PREFERABLY (although not absolutely essential) add 1ul of 1mg/ml to 20mg/ml (anything in that bracket acceptable) molecular grade glycogen: Adding glycogen as an inert carrier will increase recovery of precipitated product, particularly if 1-5ug order of magnitude and also render the precipitated pellet more visible, making washing with 70% ethanol easier. However, providing you can see the pellet which if > 1ug is possible this is not absolutely essential so you can if necessary precipitate with slat + ethanol alone
4. Take eluted PCR product plus salt + ethanol and Glycogen and incubate for 1 hour to over nigh @ -20C (if glycogen added you only need to incubate for 1 hour for efficient recovery but if convenient it wont harm to leave over night)
Spin @ 13,000rpm in an eppendorf for 15-20 min
5. Using a p200 remove most of the ethanol from the top of the aqueous layer down to the surface of the pellet. Now remove the remainder of the aqueous phase with a p20: If precipitated with glycogen the PCR pellet will be opaque white and very visible; If precipitated without glycogen the pellet will be smaller & white so hold eppendorf up to the light to identify pellet and remove aqueous phase as described
6. Now add 0.ml to 1 ml of room temp 70% ethanol
7. If glycogen added vortex the tube to detach pellet in order to more efficiently wash: The 70% ethanol wash id designed to desalt pellet; Vortexing to detach will increase desalting effectiveness and salt is more soluble in room temp ethanol (hence why you use RT ethanol and not ethanol placed at -20C)
8. If precipitated without glycogen, hold tube up to the light add ethanol gently down the sides, remove as described and add the same volume of 70% ethanol back
9. Now spin tube for 5 min at 13,000rpm in order to secure insoluble pellet to the side of the tube
10. Remove ethanol as before: This time however when you reach the level of the pellet remove the last few ul with a p10 tip
11. Now spin the tube for a further 1 min @ 13,000rpm to bring down the residual 70% ethanol up the sides of the tube. This normally amounts to a few ul
12. Remove this residual ethanol with a p10 tip
13. Now air dry the pellet for about 20 min @ room temp: When dessicated the pellet - unless contaminated with salt and protein - will turn clear, indicating it has dried
14. Now simply resuspend in a smaller volume of molecular grade water or TE (relative to starting eluted volume of PCR product) in order to make more concentrated
Good luck !!
If you have enough DNA of low concentration then you simply put this at 95 degree for some time. The water will evaporate and you will get concentrated product. This can be used in in vitro transcription or if you want to digest the DNA then also it will work but in that case you need to put it at 37 degree-55 degree for some tie to anneal it back.
Also if you are using a kit for PCR cleanup then do not use water for elution. You can use prewarmed (65 degree) elution buffer and after applying this keep it to rest fr 1-2 min. By this simple trick you will increase the yield of elution. Hope it works for you.
Dear Sad
I wouldn't suggest heating your DNA for prolonged periods, especially if in water as this can lead to damage of your DNA through depurination
However, I do accept Rams point about eluting from a colum using either molecular grade water pre heated to 65C or Tris EDTA (TE = 10mM Tris pH 8 + 0.1mM EDTA) pre heated to 65C. This is indeed an accepted trick for increasing concentration of your final eluted product
Also acknowledging and elaborating on some thing else Ram said I would and in fact do do the following:
1. Elute with 30ul volume of either water or TE and as mentioned add that volume preheated
2. Before eluting bu spinning @ 13K for 1 minute WAIT 5 minutes
3. Then take the eluate re apply to the column, wait another 5 minutes and spin again: This tends to yield the highest concentration of product
4. Alternatively, for highest yield and concentration add your 30ul of pre heated TE/water; wait 5 minutes; spin elute for 1 minute; add another 20ul of fresh water or TE to the column; wait 5 minutes; spin elute (for 1 minute) and finally pool the 2 eluates giving a total eluate fraction of 50ul
In the past when I have needed concentration exceeding 1ug/ul I have taken that 30-50ul; precipitated as described in my first answer and then re suspended in 10ul
Alternatively, you could take (and I have done this as well) your double eluted fraction and concentrate by spinning through a selective membrane
Amicon columns are probably the best known of these spin concentration fractionaters, cf.
https://www.merckmillipore.com/GB/en/product/Amicon-Ultra-0.5%C2%A0mL-Centrifugal-Filters-for-DNA-and-Protein-Purification-and-Concentration,MM_NF-C82301?bd=1
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