26 replies to this topic

[xyz]

Can you please provide me the exact protocol of it.
Thanks

swanny, on Jun 10 2009, 06:37 PM, said:

xyz, on Jun 11 2009, 03:56 AM, said:

It sound good but does it cause any problem in firther cloning assay
thanks

swanny, on Jun 9 2009, 06:52 PM, said:

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

There shouldn't be any problems at all. Once you have done the EtOH precipitation, it's as clean as DNA prepared by kits. Just make sure you get rid of trace EtOH, by leaving the tube open and inverted for a few minutes. You can also heat gently for a few minutes to make sure.

[mastermi]

xyz, on Jun 10 2009, 07:53 PM, said:

Can I do like that !!!because I have to ligate it before and for that minimum amount of DNA is required 100 ng.

mastermi, on Jun 9 2009, 01:49 PM, said:

Although you don't see anything on the gel: Just try to clone it.

And if that doesn't work: Avoid using gels and kits! Make phenol-chloroform-extraction or only ethanol-precipitation instead...

100 ng is waht the protocol says, and that is probably the best way to make sure that ligation does work.
But that doesn't mean that it doesn't work below that.
I have often ligated fragments that I haven't seen on the gel, and most times it worked out well (maybe I had just 1 out of 24 instead of 12 out of 24 right clones).
Just take less amount of your vector to make sure your vector-fragment-ratio isn't too bad.

[xyz]

Thanks a lot. what do you suggest for the vector DNA ratio.

mastermi, on Jun 11 2009, 06:30 AM, said:

xyz, on Jun 10 2009, 07:53 PM, said:

Can I do like that !!!because I have to ligate it before and for that minimum amount of DNA is required 100 ng.

mastermi, on Jun 9 2009, 01:49 PM, said:

Although you don't see anything on the gel: Just try to clone it.

And if that doesn't work: Avoid using gels and kits! Make phenol-chloroform-extraction or only ethanol-precipitation instead...

100 ng is waht the protocol says, and that is probably the best way to make sure that ligation does work.
But that doesn't mean that it doesn't work below that.
I have often ligated fragments that I haven't seen on the gel, and most times it worked out well (maybe I had just 1 out of 24 instead of 12 out of 24 right clones).
Just take less amount of your vector to make sure your vector-fragment-ratio isn't too bad.

[epigenetics]

[quote name='xyz' date='Jun 9 2009, 09:51 AM' post='26329']
Hi
Thanks for suggestion. What is the rationale for native Page( poly acrylamide GE). and one more question is that checking the conc with spec (Nanodrop) makes any sense?
[quote name='epigenetics' post='26304' date='Jun 9 2009, 06:10 AM']Just one suggestion from my side.

Well,
I was trying to digest with MBO1, bands should be of following sizes
Undigested band: 152
Digested: 125 and 27

In agarose gel, i never saw the 125 band for unknown reason, so was thinking digestion did not work, 1 week i wasted.

Then i ran 12% Native PAge which is good for 50-200 bp DNA fragments.
saw two bands, one at 152 and other at 125 in digested sample and only 152 in undigested one.

May be it is mysterious, but worked for me.

Edited by epigenetics, 11 June 2009 - 11:11 AM.

[mastermi]

xyz, on Jun 11 2009, 07:02 PM, said:

Thanks a lot. what do you suggest for the vector DNA ratio.

mastermi, on Jun 11 2009, 06:30 AM, said:

xyz, on Jun 10 2009, 07:53 PM, said:

Can I do like that !!!because I have to ligate it before and for that minimum amount of DNA is required 100 ng.

mastermi, on Jun 9 2009, 01:49 PM, said:

Although you don't see anything on the gel: Just try to clone it.

And if that doesn't work: Avoid using gels and kits! Make phenol-chloroform-extraction or only ethanol-precipitation instead...

100 ng is waht the protocol says, and that is probably the best way to make sure that ligation does work.
But that doesn't mean that it doesn't work below that.
I have often ligated fragments that I haven't seen on the gel, and most times it worked out well (maybe I had just 1 out of 24 instead of 12 out of 24 right clones).
Just take less amount of your vector to make sure your vector-fragment-ratio isn't too bad.

When I have so poor fragment, I ususally don't waste any for measuring its concentration but take all of it for ligation (elute it in the volume you can maximal put in your ligation). I then simply use 1/4 of the amount of vector I normally use. That would still be enough colonies after transformation...

[swanny]

xyz, on Jun 11 2009, 02:32 PM, said:

Can you please provide me the exact protocol of it.
Thanks

swanny, on Jun 10 2009, 06:37 PM, said:

xyz, on Jun 11 2009, 03:56 AM, said:

It sound good but does it cause any problem in firther cloning assay
thanks

swanny, on Jun 9 2009, 06:52 PM, said:

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

There shouldn't be any problems at all. Once you have done the EtOH precipitation, it's as clean as DNA prepared by kits. Just make sure you get rid of trace EtOH, by leaving the tube open and inverted for a few minutes. You can also heat gently for a few minutes to make sure.

The protocol is pretty much as I wrote above. Make sure the gel is diced into small pieces, as this speeds up the diffusion process into the TE.
The agarose is spun down hard for 10 minutes and the S/N is simply decanted off. Precipitation can be done with either sodium or ammonium acetate. If your yields are expected to be low, co-precipitate with glycogen, using standard procedures.

[xyz]

Thanks

swanny, on Jun 11 2009, 10:26 PM, said:

xyz, on Jun 11 2009, 02:32 PM, said:

Can you please provide me the exact protocol of it.
Thanks

swanny, on Jun 10 2009, 06:37 PM, said:

xyz, on Jun 11 2009, 03:56 AM, said:

It sound good but does it cause any problem in firther cloning assay
thanks

swanny, on Jun 9 2009, 06:52 PM, said:

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

There shouldn't be any problems at all. Once you have done the EtOH precipitation, it's as clean as DNA prepared by kits. Just make sure you get rid of trace EtOH, by leaving the tube open and inverted for a few minutes. You can also heat gently for a few minutes to make sure.

The protocol is pretty much as I wrote above. Make sure the gel is diced into small pieces, as this speeds up the diffusion process into the TE.
The agarose is spun down hard for 10 minutes and the S/N is simply decanted off. Precipitation can be done with either sodium or ammonium acetate. If your yields are expected to be low, co-precipitate with glycogen, using standard procedures.

[liweixie]

xyz, on Jun 8 2009, 12:37 PM, said:

Hi all,
I amplified my PCR product and it gave a nice band (4 microgram conc around), after that I puified it with qiagen kit and digested it with BAMh1 enzyme , after that I didn't get any band on gel. What could be the reason. I tried both  qiagen I and Qiaex II kit for purification but no yield. Need suggestions.

Make sure the enzyme does not have the star activity .
After you purified from gel, check the concentration,
One more thing is if your digested product is two short, you need a large amount of product if you want to see a band one the gel.

[Qundo12]

Recently I've got the same problem. After the PCR, I even didn't purify the product, used it directly for the digestion, then performed gel extraction. The amount left of digested product reduced considerably, almost lost from the gel. I have to increase the amount of PCR product 3 times before the digestion and ligation to make sure I have enough DNA.
I tried ligating when I didn't see the band before, sometimes it worked, sometimes it didn't. I agree that the most important one is the ratio. Normally, the molar ratio of 1 vector: 3 insert or 1:5 worked well for me, even when I used just 30ng of vector for 20ul ligation reaction and just transformed 3ul by electroporation.

[phage434]

If you digest a PCR product without purifying the PCR enzymes and dNTPs, then the PCR enzymes will extend the 3' end of digested ends.  This will destroy the site, and eliminate any possibility of ligating it with a vector.

[Qundo12]

phage434, on Jun 22 2009, 10:03 AM, said:

If you digest a PCR product without purifying the PCR enzymes and dNTPs, then the PCR enzymes will extend the 3' end of digested ends.  This will destroy the site, and eliminate any possibility of ligating it with a vector.

I agree that some DNA polymerases can extent 3'dA, but there are some points:
1. To some percentage of PCR products (or digested products), this reaction is succeed (in the case of Taq polymerase, about 30%).
2. After PCR, DNA pol reaches or over its half-life time (I ran 30 cycles and 30 sec at 95 degree per cycle + initiation step at 95 degree for 5-10 minutes), so the active number of enzyme molecules and their activity reduce considerably.
3. The salt condition changes when I add RE buffer.

So, this activities just "destroy" a little amount of PCR products, but I gain the higher amount of DNA if I pass the purification step.
Actually, this works for me many times  

[hanming86]

swanny, on Jun 10 2009, 05:37 PM, said:

xyz, on Jun 11 2009, 03:56 AM, said:

It sound good but does it cause any problem in firther cloning assay
thanks

swanny, on Jun 9 2009, 06:52 PM, said:

Some of the groups in my institute are moving away from kits etc completely, and just crush the gel slice, make up to 1 .5 ml with TE and leave in the fridge overnight. The next morning they spin down the agarose, remove the S/N and do an ethanol precipitation. Slow, I know, but it's faster in the long run. If the DNA product is smallish, use ammonium acetate rather than sodium acetate: 1/3 vol 7.5M ammonium Ac, 2 volumes of the new volume EtOH.

There shouldn't be any problems at all. Once you have done the EtOH precipitation, it's as clean as DNA prepared by kits. Just make sure you get rid of trace EtOH, by leaving the tube open and inverted for a few minutes. You can also heat gently for a few minutes to make sure.

EtOH precipitation probably can give you cleaner DNA than kits.