Difference: ProtocolsDpnIDigestion (1 vs. 4)

Revision 42017-11-06 - SeanLeonard

  META TOPICPARENT 
 name="ProtocolUpdatesNeeded" 

 Dpn I digestion
- META TOPICPARENT
+ name="ProtocolUpdatesNeeded"
-<
<
+Purpose
->
>
+Purpose
-<
<
+To digest (Adeno) methylated GATC sites. Useful for removing cell-derived plasmid template from PCR samples.
->
>
+To digest (Adeno) methylated GATC sites. Useful for removing cell-derived plasmid template from PCR samples.
-<
<
+Use
->
>
+Use
-<
<
+.  Add 1µL of DpnI to finished 50µL PCR reactions (or .5µL to 25µL reactions).    Pipet or invert to mix.
->
>
+. Add 1µL of DpnI to finished 50µL PCR reactions (or .5µL to 25µL reactions). Pipet or invert to mix. 
 2. Incubate the mixture at 37°C for 1-2 hrs (depending on your paranoia or need to remove template DNA). Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but is unnecessary). 
 3. PCR cleanup or gel-purify the reaction for downstream processes. It's that easy!
-<
<
+.  Incubate the mixture at 37°C for 1-2 hrs (depending on your paranoia or need to remove template DNA).  Alternatively, the solution can be left overnight at room temperature.  Periodic mixing may aid digestion (but is unnecessary). 

3.  PCR cleanup or gel-purify the reaction for downstream processes.  It's that easy!
->
>
+Other Notes
-<
<
+Other Notes
->
>
+DpnI can (and should) be added directly to PCR sample.
-<
<
+Use in NEBuffer 4; however, it can be added directly to PCR sample.
->
>
+Outside of PCR reactions, use DpnI with NEBuffer 4 or Custmart.
-<
<
+Heat inactivate by incubating at 80°C for 20 minutes.
->
>
+Heat inactivate by incubating at 80°C for 20 minutes.
-<
<
+Procurement
->
>
+Procurement
-<
<
+Can be ordered directly from NEB.
->
>
+Can be ordered directly from NEB. 
 Typically stored at -20°C; can be found in the common enzyme freezer box.
-<
<
+Typically stored at -20°C;  can be found in the common enzyme freezer box.
 NEB unit definition:

One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 DNA (dam methylated) in 1 hour at 37°C in a total reaction volume of 50 µl.

NEB buffer composition:
-<
<
+X NEBuffer 4:
->
>
+X NEBuffer 4: 
 20 mM Tris-acetate 
 50 mM potassium acetate 
 10 mM Magnesium Acetate 
 1 mM Dithiothreitol 
 pH 7.9 @ 25°C
-<
<
+mM Tris-acetate 

50 mM potassium acetate 

10 mM Magnesium Acetate 

1 mM Dithiothreitol 

pH 7.9 @ 25°C

Revision 32014-07-23 - SeanLeonard

META TOPICPARENT	name="ProtocolUpdatesNeeded"

Dpn I digestion

Purpose

To digest (Adeno) methylated GATC sites. Useful for removing cell-derived plasmid template from PCR samples.

Use

Changed:

<
<

1. Add 1µL of DpnI to 50µL PCR reactions (or .5µL to 25µL reactions). Pipet or invert to mix.

>
>

1. Add 1µL of DpnI to finished 50µL PCR reactions (or .5µL to 25µL reactions). Pipet or invert to mix.

2. Incubate the mixture at 37°C for 1-2 hrs (depending on your paranoia or need to remove template DNA). Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but is unnecessary).
3. PCR cleanup or gel-purify the reaction for downstream processes. It's that easy!

Other Notes

Use in NEBuffer 4; however, it can be added directly to PCR sample.

Heat inactivate by incubating at 80°C for 20 minutes.

Procurement

Can be ordered directly from NEB.
Typically stored at -20°C; can be found in the common enzyme freezer box.

NEB unit definition:

One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 DNA (dam methylated) in 1 hour at 37°C in a total reaction volume of 50 µl.

NEB buffer composition:

1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C

Revision 22014-07-21 - SeanLeonard

  META TOPICPARENT 
 name="ProtocolUpdatesNeeded" 

 Dpn I digestion 

Purpose 


To digest (Adeno) methylated GATC sites. Useful for removing cell-derived plasmid template from PCR samples.
- META TOPICPARENT
+ name="ProtocolUpdatesNeeded"
->
>
+Use
-<
<
+Procurement
->
>
+.  Add 1µL of DpnI to 50µL PCR reactions (or .5µL to 25µL reactions).    Pipet or invert to mix.
->
>
+.  Incubate the mixture at 37°C for 1-2 hrs (depending on your paranoia or need to remove template DNA).  Alternatively, the solution can be left overnight at room temperature.  Periodic mixing may aid digestion (but is unnecessary). 

3.  PCR cleanup or gel-purify the reaction for downstream processes.  It's that easy!
-<
<
+Can be ordered directly from NEB. 

Typically stored at -20°C;  can be found in the common enzyme freezer box.
->
>
+Other Notes
-<
<
+Use
 Use in NEBuffer 4; however, it can be added directly to PCR sample.
-<
<
+Incubate at 37°C for one hour or overnight.
 Heat inactivate by incubating at 80°C for 20 minutes.
->
>
+Procurement 


Can be ordered directly from NEB. 

Typically stored at -20°C;  can be found in the common enzyme freezer box.
 NEB unit definition:

One unit is defined as the amount of enzyme required to digest 1 µg of pBR322 DNA (dam methylated) in 1 hour at 37°C in a total reaction volume of 50 µl.

NEB buffer composition:

1X NEBuffer 4: 

20 mM Tris-acetate 

50 mM potassium acetate 

10 mM Magnesium Acetate 

1 mM Dithiothreitol 

pH 7.9 @ 25°C

Revision 12012-08-15 - CraigBarnhart

META TOPICPARENT	name="ProtocolUpdatesNeeded"