b******r
发帖数: 111 | 1 These days, almost make me desperate to death. As a 2-year post-doc,who did
lots of cloning, I fail to do such an easy work. Isolate mouse genomic DNA
by phenol/chloroform,75%ethanol precipitation. Then digest by HindIII
restriction enzyme as follows:
genomic DNA: 2 ug,1ug,and 0.4ug;
HindIII enzyme: 70 units(3.5ul,and different units);
Buffer: 5 ul
add water to 50ul
37 degree for 10 hours. Run gel.Genomic DNA is still a strong band, not
smear at all. I have try HindIII from NEB and Roche companies. Both don't
work. But cut plasmid very well. Any one can tell me how to fix this
problem? It takes me one week to try any ideas I could think out. Please
help me out. My mentor is angry to me again and again. Or else, I think I
have to leave this lab. | s******s
发帖数: 13035 | 2 你试一下column based的方法抽gDNA吧。我怀疑是不是蛋白没除尽，或者
有phenol残余。
did
not
【在 b******r 的大作中提到】
: These days, almost make me desperate to death. As a 2-year post-doc,who did
: lots of cloning, I fail to do such an easy work. Isolate mouse genomic DNA
: by phenol/chloroform,75%ethanol precipitation. Then digest by HindIII
: restriction enzyme as follows:
: genomic DNA: 2 ug,1ug,and 0.4ug;
: HindIII enzyme: 70 units(3.5ul,and different units);
: Buffer: 5 ul
: add water to 50ul
: 37 degree for 10 hours. Run gel.Genomic DNA is still a strong band, not
: smear at all. I have try HindIII from NEB and Roche companies. Both don't
| h******y
发帖数: 351 | 3 1. 你抽提的DNA有问题。
2. 试试其他酶，例如EcoRI
by the way, 你用的酶量太大。
did
not
【在 b******r 的大作中提到】
: These days, almost make me desperate to death. As a 2-year post-doc,who did
: lots of cloning, I fail to do such an easy work. Isolate mouse genomic DNA
: by phenol/chloroform,75%ethanol precipitation. Then digest by HindIII
: restriction enzyme as follows:
: genomic DNA: 2 ug,1ug,and 0.4ug;
: HindIII enzyme: 70 units(3.5ul,and different units);
: Buffer: 5 ul
: add water to 50ul
: 37 degree for 10 hours. Run gel.Genomic DNA is still a strong band, not
: smear at all. I have try HindIII from NEB and Roche companies. Both don't
| g*****n
发帖数: 250 | 4 有phenol残余的可能性大。用chloroform 再洗一次，再沉淀。 | b******r
发帖数: 111 | 5 Thanks. Can you tell me the name of 'column based' purification of genomic
DNA?
【在 s******s 的大作中提到】
: 你试一下column based的方法抽gDNA吧。我怀疑是不是蛋白没除尽，或者
: 有phenol残余。
:
: did
: not
| b******r
发帖数: 111 | 6 I have done '用chloroform 再洗一次，75% ethanol 再沉淀。',but yield is too
low.
Can I do this:
add NaAc(0.1 volume of genomic DNA), mix,then 2.5 volume of 100% ethanol to
precipitate again? The question is: does a little NaAc still stay in
purified genomic DNA and inhibit digestion of HindIII?
【在 g*****n 的大作中提到】
: 有phenol残余的可能性大。用chloroform 再洗一次，再沉淀。
| g*****n
发帖数: 250 | 7 The question is: does a little NaAc still stay in
purified genomic DNA and inhibit digestion of HindIII?
Maybe. You can use a large volume (e.g., 0.5 ml) of 70% ethanol to wash the
DNA pellet. | a****d
发帖数: 1919 | 8 like this one?
DNeasy Blood & Tissue Kit
http://www.qiagen.com/products/genomicdnastabilizationpurificat
【在 b******r 的大作中提到】
: Thanks. Can you tell me the name of 'column based' purification of genomic
: DNA?
| b******r
发帖数: 111 | |
|
