Digests: Difference between revisions

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=== Protocol ===
{| class="wikitable"
|'''Protocol'''
|}
1. Check the NEB (or other supplier) website to confirm enzyme compatibility
 
2. In a PCR tube add the reaction components according to the table listed below
 
3. Mix contents gently and spin down using the small benchtop centrifuge


Add the following reagents into a round-lidded PCR tubes:<br>
3. Incubate at 37°C for 30 min if time-saver qualified, 60 minutes if not


{| cellspacing="1" cellpadding="1" border="1" style="width: 506px; height: 142px;"
{| class="wikitable"
|'''Example  Reaction                                                                                                                              '''
|}
{| class="wikitable"
|'''Component'''
|'''Volume'''
|-
|-
! scope="col" | Reagents<br>
|ddH2O
! scope="col" | PCR insert (µL)<br>
|Up to 50 µL
! scope="col" | Plasmid (µL)<br>
|-
|-
| DNA<br>
|10X rCutSmart Buffer
| 50<br>
|5 µL
| X (~3-5 µg)<br>
|-
|-
| FD 10x buffer<br>
|DNA
| 5.6<br>
|x (1 µg)
| 2<br>
|-
|-
| Restriction enzyme<br>
|Enzyme 1
| 1<br>
|1 µL
| 1<br>
|-
|-
| ddH<sub>2</sub>O<br>
|Enzyme 2
| 0<br>
|1 µL
| up to 20 µL<br>
|}
|-
{| class="wikitable"
| Total<br>
|'''Notes'''
| 56.6<br>
| 20<br>
|}
|}
-         NEB High-Fidelity (HF) enzymes are ordered whenever possible, they all use rCutSmart reaction buffer and are usually time-saver qualified Hi


#Mix tube(s) gently by tapping.
-         NEB has the option to select your specific enzymes and generate a detailed protocol
#Centrifuge for 1 minute at 4,500 xg.
#Incubate tube(s) for 2 hours at 37°C.
#Heat inactivate at 65°C (or 80°C, depending on the restriction enzyme(s) used) for 20 minutes.The heat inactivation temperature for the restriction enzymes can be found in the protocol folder or online, and for double digests, the larger of the two heat inactivation temperatures should be used.<br>
#Cool tube(s) on ice for 2 minutes.
#Add 1 µL of Calf Intestinal Phosphatase (CIP) and X µL of 10x NEB buffer to the ''plasmid'' tube. Note: CIP is not necessary for double digests, and is only necessary when planning on following the digest with a ligation.<br>
#Incubate the plasmid tube at 37°C for 1 hour. The tube with the insert may remain on ice.
#Run both the plasmid and insert digests out on an agarose gel. Note: to reduce the risk of cross-contamination, ensure that there is ample space between the samples.
#After the gel has run, cut out the desired bands using UV light and place the bands into new and separate microcentrifuge tubes.
#Purify the samples using the Geneclean II kit. Note: If the amount of water in the glassmilk container is low, add enough ddH<sub>2</sub>O that the ratio of liquid to solid is approximately 1:1. Also, in step 12 of protocol 5.1 (purifying from solutions) and step 18 of protocol 5.2 (purifying from agarose gel) use 13 µL of ddH<sub>2</sub>O to extract the DNA.<br>
#Quanitfy DNA samples. Note: a good yield is ~30-150 ng/µL.<br>


<br>
-         Heat inactivation may be recommended if there are additional steps in the workflow without a DNA purification step
 
{| class="wikitable"
|'''References, related resources, and acknowledgments'''
|}
-         <nowiki>https://nebcloner.neb.com/#!/redigest</nowiki>

Revision as of 18:40, 17 January 2025

Protocol

1. Check the NEB (or other supplier) website to confirm enzyme compatibility

2. In a PCR tube add the reaction components according to the table listed below

3. Mix contents gently and spin down using the small benchtop centrifuge

3. Incubate at 37°C for 30 min if time-saver qualified, 60 minutes if not

Example Reaction                                                                                                                              
Component Volume
ddH2O Up to 50 µL
10X rCutSmart Buffer 5 µL
DNA x (1 µg)
Enzyme 1 1 µL
Enzyme 2 1 µL
Notes

-         NEB High-Fidelity (HF) enzymes are ordered whenever possible, they all use rCutSmart reaction buffer and are usually time-saver qualified Hi

-         NEB has the option to select your specific enzymes and generate a detailed protocol

-         Heat inactivation may be recommended if there are additional steps in the workflow without a DNA purification step

References, related resources, and acknowledgments

-         https://nebcloner.neb.com/#!/redigest

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