digestion with two REs requiring different buffer - (Oct/06/2005 )

Hello, everyone,

I need to digest my plasmid with HindIII and BclI but HindIII requires low salt buffer(50 mM NaCl) and Bcl1 high salt buffere(100mM NaCl).

How can I do that??


thanks ahead,

forforfor

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digest with hindIII first then dilute 1:1 with a master mix containing 2X Bcl1 buffer and enzyme and incubate again.

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Sequential digest: First digest with HindIII, then bump up the salt concentration with NaCl to 100mM while adding H2O and the buffer for that enzyme to compensate for the increased volume. Add the second enzyme and digest and you should be good to go.

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thanks. I am slow with math. Let's say the digestion with HindIII is 50 ul total, how much 10X BCl1 buffere and how much Bcl 1 enzyme I should add ? how much is the total reaction for the second incubation?

one more question, do I digest with hindIII overnight first and do Bcl1 the next day??

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thanks. Can you tell me why need to add H2O and buffer for the enzyme since the salt concentration is already brought up??

forforfor

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Where did you buy your enzyme? Have you checked the activity of both enzymes in various other buffers? For example, New England Biolabs has this data for HindIII and BclI:

HindIII
Activity in NEBuffers:
NEBuffer 1: 50%
NEBuffer 2: 100%
NEBuffer 3: 10%
NEBuffer 4: 50%

BclI
Activity in NEBuffers:
NEBuffer 1: 50%
NEBuffer 2: 100%
NEBuffer 3: 100%
NEBuffer 4: 75%

1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM dithiothreitol
pH 7.9 @ 25°C

So, even though HindIII from this vendor ships with their buffer 2, and BclI from them ships with buffer 3, a double digest using buffer 2 should work wonderfully...

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Facing few recent topics on that, i would remind you that Bcl I is a dam sensitive enzyme...
Homebrew is right. you'll get often enzymes that shows 100%cut in more than 1 buffer. and if you only look at the recommended one, you may pass over the info.
For your digestion, i would complement the information iven by homebrew by mention tat you first should digest with HindIII for at least 2h, then increase temperature to 50° (optimal for Bcl I) and add 1µl of enzyme in your prep, and let stay for at least 2 more hours.

Fred

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Ah -- I didn't notice BclI was a 50° enzyme. Thanks for picking that up, fred. Fred's also right on about the appropriately-named dam methylation -- there's some info on that here.

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thanks to all.

My hindIII is from invitrogen but Bcl1 from New England Biolabs.

and two more questions

1. what's the difference between digesting 2 hours and over night? is that the longer the better? when I did my cloning, I was suggested to digest my foreign DNA for 1 hour but plasmid for overnight? Why is that?

2. for 1ug DNA , how many units of RE is normally recommended? I didn't see specific information on NEB's catalogue.


good weekend,

forforfor

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hi
NEB ensures that 100% is possible to get in 2h at recommended temperature. But sometimes, digesting overnight allows you to get complete digestion if your prep is not that clean. Sometimes it allows to save time.
But in this case you should take care of the star activity (degradation whithout any target sequence, neb web site gives some tips on enzymes, and you can access enzyme by enzyme, which one is suspected to have star activity).
if enzyme shows a star activity, i do my digestion in a PCR tube with a program :
optimal temp for 4h
hold 4°C

from neb web site :

for 1µg DNA, 5units (0.5 µl is enough) for 2h digestion.

good week end to you too.
fred

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