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Can someone help me with my test question regarding ligation mixture?


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5 replies to this topic

#1 Beatrix Chin

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Posted 06 January 2015 - 09:00 AM

Prepare a reaction mix for a restriction digest containing components from the following stock solution : 

Plasmid DNA (2mg/ml) 
Reaction buffer (10X) 
BamH1 (10U/ul) 

Indicate the minimum total reaction volume required for digesting 10 ug of plasmid DNA and the order in which these reagents should be added.



#2 bob1

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Posted 06 January 2015 - 01:01 PM

Moved because this is a homework question.  Also because it is homework - you should provide some input as to what you think and why, so that we can help you learn.



#3 Beatrix Chin

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Posted 06 January 2015 - 07:37 PM

Sorry about that. Here's my input 

By definition :
1 unit = 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl

 

the question ask for 10 ug, so i assume the total volume would be 500 ul ? correct ? 

my guess would be
- 10U of restriction enzyme (take 1 ul from stock solution)
- 50 ul of buffer (10% from total volume)
- 5 ul of plasmid DNA (contain 10 ug)
- 444 ul distilled water 

Please enlighten me. thanks smile.png
 
 



#4 bob1

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Posted 07 January 2015 - 01:10 AM

500 ul is a big reaction. You can usually digest more in smaller volumes, typical reactions are usually done in 50 ul no matter how much DNA you have. However, the glycerol in the restriction enzyme can inhibit the digestion if you have too much. I would suggest 2 x 50 ul digests with 5 ug in each.  Digests are usually done with a 2-4 fold excess of RE (e.g. 2 U per ug), but should still work with 1 U per ug. Your other calculations are correct for the volume you suggested and can be easily applied to any other volume.



#5 Trof

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Posted 07 January 2015 - 10:09 AM

Yet the question asks for the minimum volume required (which may not be ideal volume if someone has an actual experience).

 

This is tricky, because lower volumes does often work worse than bigger for reasons not easily explained nad calculated in some weird test questions.

 

You need 5 ul of plasmid. You need amount of BamHI that will cut it (lets assume it could be 1U/1ug of DNA, though this is in reality something not easily predictable, since some enzymes cut better than others, can withstand longer cutting times etc.), so 1 ul.

The less than 10% of glycerol in areaction mix is a rule, so with 1 ul of enzyme, you need at least 10 ul reaction volume, but, that would mean only 4 ul of water (1 ul of buffer) and given the concentration of DNA and the fact that you cut a huge amount of plasmid, it won't probably work, in reality. Too thick.

Also 10 ul restriction reaction is a bad idea anyway, it evaporates too quicky in 37, and so, some use 20 ul as absolute minimum for any amount, some even don't go below 50ul. (I had a reaction that was very badly cut in 20 ul, but worked nicely in 50ul, nothing else changed)

If I wanted to go really to a minumum, that I THINK may work fine, I would put it all in 50 ul reaction volume. But, if it would work or not, or if it actually will cut 100 % in a 20ul reaction as well, who knows (it also depends a lot on the impurities in the DNA and other things).

 

To sum it up, if it's not obvious enough, I personally think it is a stupid test question, using unusually high plasmid amoutn to cut, forcing you to answer a "minimal volume required" which is not the same as the "most efficient" or whatever REAL reason we may have in mind when actually making such reaction (and there may be many, depending on what do you plan to do with the plasmid afterwards).


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#6 mdfenko

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Posted 08 January 2015 - 04:20 AM

unless the teacher had given them an "ideal" concentration of dna for the reaction.


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