Purification of small DNA molecules - (Jul/06/2005 )
I have to digest 43 bp CPR amplicon with two restriction enzymes and purify the middle portion of ~25 bp fragment. I can run a 20% PAGE gel and can separate this 25 bp band from the other smaller digestion products.
As 25 bp is too small for any Qiagen column purification method, I need to find out a good method to purify this 25 bp band out of PAGE without loosing too much of it. I try to use the DNA for cloning purpose (library generation).
Anybody has a suggestion in this case?
Thanks,
SH
As 25 bp is too small for any Qiagen column purification method, I need to find out a good method to purify this 25 bp band out of PAGE without loosing too much of it. I try to use the DNA for cloning purpose (library generation).
Anybody has a suggestion in this case?
Thanks,
SH
http://axon.med.harvard.edu/~cepko/protocol/mike/D5.html
Solutions
Crush and Soak Solution
500 mM NH4OAc 3.3 g NH4OAc
0.1% SDS 0.1 g SDS
0.1 mM EDTA 20 ml 500 mM EDTA
up to 100 ml with Q
store at room temperature
3 M NaOAc pH 5.2
24.6 g anhydrous sodium acetate
pH to 5.2 with acetic acid and bring up to 100 ml with Q
store at room temperature
• Run a 4-6% acrylamide gel in 1X TBE, stain in EthBr (1-10 mg/ml) and cut out the desired band.
• Crush the acrylamide with a p1000 tip with a melted end to resemble a pestle for the eppendorf "mortar."
• Add 1 ml crush and soak solution and incubate overnight at 37° C.
• Spin in the microfuge for 10 minutes at 14,000 rpm. Remove as much liquid as possible and add another 500 microliters of crush and soak solution.
• Repeat the spin and pool the recovered supernatant.
• Add 0.1 volume of 3M NaOAc, 2.5 volumes of EtOH and carrier (glycogen or tRNA ).
• Spin as usual, wash and dry. Resuspend in 20 microliters TE.
As 25 bp is too small for any Qiagen column purification method, I need to find out a good method to purify this 25 bp band out of PAGE without loosing too much of it. I try to use the DNA for cloning purpose (library generation).
Anybody has a suggestion in this case?
Thanks,
SH
http://axon.med.harvard.edu/~cepko/protocol/mike/D5.html
Solutions
Crush and Soak Solution
500 mM NH4OAc 3.3 g NH4OAc
0.1% SDS 0.1 g SDS
0.1 mM EDTA 20 ml 500 mM EDTA
up to 100 ml with Q
store at room temperature
3 M NaOAc pH 5.2
24.6 g anhydrous sodium acetate
pH to 5.2 with acetic acid and bring up to 100 ml with Q
store at room temperature
• Run a 4-6% acrylamide gel in 1X TBE, stain in EthBr (1-10 mg/ml) and cut out the desired band.
• Crush the acrylamide with a p1000 tip with a melted end to resemble a pestle for the eppendorf "mortar."
• Add 1 ml crush and soak solution and incubate overnight at 37° C.
• Spin in the microfuge for 10 minutes at 14,000 rpm. Remove as much liquid as possible and add another 500 microliters of crush and soak solution.
• Repeat the spin and pool the recovered supernatant.
• Add 0.1 volume of 3M NaOAc, 2.5 volumes of EtOH and carrier (glycogen or tRNA ).
• Spin as usual, wash and dry. Resuspend in 20 microliters TE.
Thank you.
The problem is because I heard that DNA less than 50 bp will be lost during Ethanol precipitation...??
The use of yeast tRNA as a carrier for ethanol precipitation of DNA carries an inherent risk of cloning yeast genomic DNA fragments that frequently contaminate tRNA preps. In addition, excess tRNA inhibits ligation/transformation. Glycogen is safer, but since it is isolated from mussels, it also carries the risk of contamination by DNA.
0.5% Linear PolyAcrylamide (LPA) is a completely inert carrier used for ethanol precipitations : Prepare a 5% polyacrylamide solution in 40 mM Tris-HCl, pH 7.8, 20 mM sodium acetate, 1 mM EDTA and polymerize with 0.1% APS and 1/1000 volume TEMED. In a 50 mL Falcon 2098 centrifuge tube, mix in the order listed: 0.25 g acrylamide (no bis-acrylamide) with 4.25 mL of Type I water, 200 µL of 1 M Tris-HCl, pH 8.0, 33 µL of 3 M sodium acetate, pH 7.5, 10 µL of 0.5 M EDTA and 50 µL of 10% APS. Add 5 µL of TEMED and mix well to begin polymerization. Incubate at room temperature for 30 minutes. Add 12.5 mL of 100% ethanol and mix by inversion to cause immediate heavy precipitation. 3-5 minutes of gentle inversion should gradually create a large white egg-shaped ball of precipitated polyacrylamide. Centrifugation is not required. Pour off the supernatant and wash with 10 mL of room temperature 70% ethanol. Mix by inversion and carefully decant the ethanol. Use a pipettor to remove residual ethanol and let the “pellet” air dry briefly. Complete evaporation of the ethanol is not required, as this solution is destined for ethanol precipitations. Resuspend the pellet to the 50 mL mark of the Falcon tube with Type I water. Allow the pellet to dissolve overnight. Mix briefly and prepare 1 mL aliquots of 0.5% LPA and store at 4°C (stable over 1 year). (Adapted from Gaillard, C. and Strauss, F. (1990) Nucleic Acids Res. 18, 378.)
0.5% Linear PolyAcrylamide (LPA) is a completely inert carrier used for ethanol precipitations : Prepare a 5% polyacrylamide solution in 40 mM Tris-HCl, pH 7.8, 20 mM sodium acetate, 1 mM EDTA and polymerize with 0.1% APS and 1/1000 volume TEMED. In a 50 mL Falcon 2098 centrifuge tube, mix in the order listed: 0.25 g acrylamide (no bis-acrylamide) with 4.25 mL of Type I water, 200 µL of 1 M Tris-HCl, pH 8.0, 33 µL of 3 M sodium acetate, pH 7.5, 10 µL of 0.5 M EDTA and 50 µL of 10% APS. Add 5 µL of TEMED and mix well to begin polymerization. Incubate at room temperature for 30 minutes. Add 12.5 mL of 100% ethanol and mix by inversion to cause immediate heavy precipitation. 3-5 minutes of gentle inversion should gradually create a large white egg-shaped ball of precipitated polyacrylamide. Centrifugation is not required. Pour off the supernatant and wash with 10 mL of room temperature 70% ethanol. Mix by inversion and carefully decant the ethanol. Use a pipettor to remove residual ethanol and let the “pellet” air dry briefly. Complete evaporation of the ethanol is not required, as this solution is destined for ethanol precipitations. Resuspend the pellet to the 50 mL mark of the Falcon tube with Type I water. Allow the pellet to dissolve overnight. Mix briefly and prepare 1 mL aliquots of 0.5% LPA and store at 4°C (stable over 1 year). (Adapted from Gaillard, C. and Strauss, F. (1990) Nucleic Acids Res. 18, 378.)
As 25 bp is too small for any Qiagen column purification method, I need to find out a good method to purify this 25 bp band out of PAGE without loosing too much of it. I try to use the DNA for cloning purpose (library generation).
Anybody has a suggestion in this case?
Thanks,
SH
I wonder if 0.5% Linear PolyAcrylamide (LPA) will or will nor interfere with downstream applications like restriction digestions or ligation?