Hi
I used to use mini-spin columns of Qiagen for my purifications, but they are too expensive for frequently usage. Now it's about 2 month that I use preparative gel electrophoresis and excise my band, this method is so time consuming, do you know a simpler and qiuker way?
I usually use purification of my products after:
1-PCR
2-Digestion with restriction enzymes which later I want to use them for ligation
3-Dephosphorilation with CIP, and then ligation
I want to know if basicaly purification is essential for 2 & 3???
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3 replies to this topic
#2
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Posted 24 March 2005 - 08:51 AM
Again Me!
Could you also tell me how do you gel extract your products?
I first do gel electerophoresis in big wells and then cut the band ( it's normal gel, not lowmwlting) and after that I freez the gel in liquid nitrogen and then setrifuge...,
do you use better way?
Could you also tell me how do you gel extract your products?
I first do gel electerophoresis in big wells and then cut the band ( it's normal gel, not lowmwlting) and after that I freez the gel in liquid nitrogen and then setrifuge...,
do you use better way?
#3
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Posted 24 March 2005 - 09:05 AM
hi
for PCR, methylnick suggests in this topic:
for your digestions, if you do not physically separate insert /plasmid, you'll get plenty of re ligation!!
dephopshorylation with cip :
if it's from a purified plasmid, ethanol/na acetate will do ok
if it's digestion followed by dephosphorylation, it's the same problem than upper.
as methylnick told us a quick and easy way to get dna from agarose gel, why don't you try it?
fred
for PCR, methylnick suggests in this topic:
Quote
I can suggest a cheaper alturnative. And you would be sure to not lose the product because it may not bind to the column.
You can run your PCR on a agarose gel, cut the band of interest out, place into an eppy and add 100uL of TE. Centrifuge to ensure the slice of gel is in TE, or you can now mash the gel up with a pipette tip. Then place the tube into a 60C heat block for about 15 minutes. Then spin the tube at maximum on a table top centrifuge for 20 minutes. Aspirate the supernatant which now contains only the DNA of interest. smile.gif
Good luck!
Nick
You can run your PCR on a agarose gel, cut the band of interest out, place into an eppy and add 100uL of TE. Centrifuge to ensure the slice of gel is in TE, or you can now mash the gel up with a pipette tip. Then place the tube into a 60C heat block for about 15 minutes. Then spin the tube at maximum on a table top centrifuge for 20 minutes. Aspirate the supernatant which now contains only the DNA of interest. smile.gif
Good luck!
Nick
for your digestions, if you do not physically separate insert /plasmid, you'll get plenty of re ligation!!
dephopshorylation with cip :
if it's from a purified plasmid, ethanol/na acetate will do ok
if it's digestion followed by dephosphorylation, it's the same problem than upper.
as methylnick told us a quick and easy way to get dna from agarose gel, why don't you try it?
fred
#4
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Posted 29 March 2005 - 03:12 AM
hi
exactly, i always purify by pcr products by EtOH ppt before restriction digesion. This mtd also work for plasmid for dephosphorylation. Gell purification is not necessary unless you have to isolate specific band from a mixture of bands. good luck
chandima
exactly, i always purify by pcr products by EtOH ppt before restriction digesion. This mtd also work for plasmid for dephosphorylation. Gell purification is not necessary unless you have to isolate specific band from a mixture of bands. good luck
chandima
