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Restriction digestion


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6 replies to this topic

#1 bluebird

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Posted 18 January 2010 - 03:38 PM

Hi,

I want to do restrition digest to my protein with nde 1 and Bam H 1, the protocol that I have said I shold keep them for an hour, my question is do you think an hour is sufficent and what do you expect to see in my gel how many bands ?

#2 leelee

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Posted 18 January 2010 - 11:09 PM

BamHI and NdeI are both endonucleases meaning that they will cut only DNA and not protein, but I'm assuming that was a typo.

We can't possibly tell you how many bands you will expect to see, you haven't given any information as to what you are cutting (plasmid??, PCR fragment??) and how many of each restriction sites there are or how far apart they are located.
You need to have a look at your sequence and determine where the cut sites are, and from that you will be able to see how big your fragments will be.

One hour incubation is more than sufficient for single digests with each enzyme, however if you are doing a double digest (both enzymes at the same time) you may need to increase your incubation time- see this NEB page for more info on this double digest.

http://www.neb.com/n...tCalculator.asp

#3 bluebird

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Posted 23 January 2010 - 11:19 AM

BamHI and NdeI are both endonucleases meaning that they will cut only DNA and not protein, but I'm assuming that was a typo.

We can't possibly tell you how many bands you will expect to see, you haven't given any information as to what you are cutting (plasmid??, PCR fragment??) and how many of each restriction sites there are or how far apart they are located.
You need to have a look at your sequence and determine where the cut sites are, and from that you will be able to see how big your fragments will be.

One hour incubation is more than sufficient for single digests with each enzyme, however if you are doing a double digest (both enzymes at the same time) you may need to increase your incubation time- see this NEB page for more info on this double digest.

http://www.neb.com/n...tCalculator.asp


thanks for reply. my reagents are from promega, and am digesting a plasmid iserted in vector pET-16b.
it is my firsyt time to deal with that and I want to know how the band appear looks in both cases the negative and positive digestion.

#4 leelee

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Posted 24 January 2010 - 06:26 PM

You need to look at the sequences of your vector and your vector+insert and find the restriction sites. This will tell you the size of the fragments that you should expect in either case.

Can you ask your supervisor to go through this with you? It seems strange that you would be needing advice on a basic concept such as this on a forum- are you getting enough help from your supervisor? Is there somebody else in the lab that you can have go over this with you?
You should never be afraid to ask questions- after all, it is the only way that we can learn!!

#5 bluebird

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Posted 25 January 2010 - 07:17 AM

You need to look at the sequences of your vector and your vector+insert and find the restriction sites. This will tell you the size of the fragments that you should expect in either case.

Can you ask your supervisor to go through this with you? It seems strange that you would be needing advice on a basic concept such as this on a forum- are you getting enough help from your supervisor? Is there somebody else in the lab that you can have go over this with you?
You should never be afraid to ask questions- after all, it is the only way that we can learn!!


thank you leelee for your advice. Yes you're right it is basics for biologist or science graduate but for medical graduates it is little bit unfamiliar with, I am doing a biology degree beside my professional degree and my supervisor is the worst, even he doesn't have a technician to explain or give some help,however, thank you for your time.

Edited by bluebird, 25 January 2010 - 07:18 AM.


#6 Andy Lane

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Posted 25 January 2010 - 05:10 PM

You need to look at the sequences of your vector and your vector+insert and find the restriction sites. This will tell you the size of the fragments that you should expect in either case.

Can you ask your supervisor to go through this with you? It seems strange that you would be needing advice on a basic concept such as this on a forum- are you getting enough help from your supervisor? Is there somebody else in the lab that you can have go over this with you?
You should never be afraid to ask questions- after all, it is the only way that we can learn!!


thank you leelee for your advice. Yes you're right it is basics for biologist or science graduate but for medical graduates it is little bit unfamiliar with, I am doing a biology degree beside my professional degree and my supervisor is the worst, even he doesn't have a technician to explain or give some help,however, thank you for your time.


That's not acceptable of your supervisor. S/he can't expect you to do this stuff alone. I'd be really annoyed, if I were you. I'd look up one of your basic bio textbooks about restriction enzymes, first.

#7 bluebird

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Posted 25 January 2010 - 08:23 PM

You need to look at the sequences of your vector and your vector+insert and find the restriction sites. This will tell you the size of the fragments that you should expect in either case.

Can you ask your supervisor to go through this with you? It seems strange that you would be needing advice on a basic concept such as this on a forum- are you getting enough help from your supervisor? Is there somebody else in the lab that you can have go over this with you?
You should never be afraid to ask questions- after all, it is the only way that we can learn!!


thank you leelee for your advice. Yes you're right it is basics for biologist or science graduate but for medical graduates it is little bit unfamiliar with, I am doing a biology degree beside my professional degree and my supervisor is the worst, even he doesn't have a technician to explain or give some help,however, thank you for your time.


That's not acceptable of your supervisor. S/he can't expect you to do this stuff alone. I'd be really annoyed, if I were you. I'd look up one of your basic bio textbooks about restriction enzymes, first.


I know that I will see two bands in my digestion, but I am seeing 3 thin bands, so thats why I am asking the people here,at the begining I sused homemade reagents and I thought that was the problem, then I used qiagen plasmid minirep and the problem still!!!!!, I am afraid that there is something with the plasmid itself, because it is 11 years old, and the cells that I use too is very old!!and am sorry if I disturb you with my selly questions.

Edited by bluebird, 25 January 2010 - 08:30 PM.





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