Digest and Ligation: Difference between revisions

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''The protocols for digest and ligation are from New England Biolab's "NEB Cloner" tool:'' http://nebcloner.neb.com/#!/redigest.
__FORCETOC__


'''Digestion: (1.5hrs)'''
==Goal==
*Goal: To insert a fragment of DNA into a plasmid using restriction enzymes and inserts.


Prior to digesting DNA, be sure to purify the DNA amplified with Q5 polymerase to remove excess primers. Do this with the Zymogen DNA Clean and Concentrator Kits above the microwave.  
==Pre-Protocol Questions==
1. Are you familiar with the NEBioCalculator and NEB Ligation Protocol? If not, refer to the links below.


Beforehand, turn on 37 degrees water bath and 65 degrees (for ligation heat inactivation). Calculate how much DNA you will need to add in order to digest 1ug. You can store this DNA in the -20 freezer for later use.  
''The protocols for digest and ligation are from New England Biolab's "NEB Cloner" tool:'' http://nebcloner.neb.com/#!/redigest. ''and NEBioCalculator:''"https://nebiocalculator.neb.com/#!/ligation ''and NEB Ligation Protocol with T4 Ligase:'' https://www.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202.


#Go to the cloner tool link: http://nebcloner.neb.com/#!/redigest.
==Precautions==
#Simply select your digestion enzymes from dropdown menu for either a single or double restriction enzyme digestion. The buffer efficiency for each of the enzymes will be listed for the available NEB buffer options.
* high DNA concentrations may result in an incomplete digest.
##Be aware of star* activity next to listed efficiency, indicating that the buffer may alter specificity of restriction enzymes. See baseball card binder for more information on star activity between pairs of restriction enzymes and their most efficient buffers.
#Click on "Detailed Protocol" at the top middle of the page to see reagents and corresponding volumes to add to each reaction.


Incubate reactions in a 1.5mL microcentrifuge tube for at least 1 hour.
==Restriction Enzymes in the MEE Lab (and Buffer to Use)==
* Acc651 (3.1 Buffer)
* BamHI-HF (CutSmart Buffer)
* BglII (3.1 Buffer)
* BsaI-HF (CutSmart Buffer)
* BsrGI-HF (CutSmart Buffer)
* DraI (CutSmart Buffer)
* EcoRI-HF (CutSmart Buffer)
* EcoRV-HF (CutSmart Buffer)
* FspI (CutSmart Buffer)
* HindIII-HF (CutSmart Buffer)
* KpnI-HF (CutSmart Buffer)
* MluI-HF (CutSmart Buffer)
* NcoI-HF (CutSmart Buffer)
* NdeI (CutSmart Buffer)
* NheI-HF (CutSmart Buffer)
* PstI-HF (CutSmart Buffer)
* PvuI-HF (CutSmart Buffer)
* PvuII-HF (CutSmart Buffer)
* SacI-HF (CutSmart Buffer)
* SalI-HF (CutSmart Buffer)
* SnaBI (CutSmart Buffer)
* SphI-HF (CutSmart Buffer)
* SspI-HF (CutSmart Buffer)
* XhoI (CutSmart Buffer)


Between the digestion and ligation steps, '''purify (with the DNA Clean and Concentrator Kit) both the insert and plasmid vector DNA to remove restriction enzymes'''.
==Digestion==


'''Ligation: (0.5hrs)'''
1. Prior to digesting DNA, purify DNA amplified via PCR to remove primers and to change the buffer.
:a. Do this with the Zymogen DNA Clean and Concentrator Kits above the microwave.
 
2. Beforehand, turn on 37 degrees water bath (this is for the incubation period) and dry bath at 80 degrees (if you are heat inactivating the restriction enzymes).
 
3. Answer these questions:
:a. What volume of DNA you will need to add in order to digest 1 µg?
:b. Are you digesting with multiple restriction enzymes that require different buffers? If so, please talk to Eric.
:c. Are you stopping here overnight (or longer)? Then, after the digestion, heat inactivate the enzymes at 80 degrees for 20 minutes.
 
4.
 
For a 50 µl reaction:
 
{|
|DNA||1 µg
|-
|10X Buffer (CutSmart or 3.1, depending on the enzyme)||5 µl (1X)
|-
|Each enzyme||1.0 µl
|-
|Nuclease-free Water||to 50 µl
|}
 
OR
 
For a 10 µl reaction:
 
{|
|DNA||150 ng
|-
|10X Buffer (CutSmart or 3.1, depending on the enzyme)||1 µl (1X)
|-
|Each enzyme||0.2 µl
|-
|Nuclease-free Water||to 10 µl
|}
 
5. Incubate reactions in a 1.5mL microcentrifuge tube at 37 degrees for at least 1 hour.
 
6. If necessary, store this DNA in the -20 freezer or in the 4 degree refrigerator for later use.
 
7. Purify (with the DNA Clean and Concentrator Kit) both the insert and plasmid vector DNA separately to remove the restriction enzymes.
 
==Ligation==


''Navigate to the NEBioCalculator:'' https://nebiocalculator.neb.com/#!/ligation .
''Navigate to the NEBioCalculator:'' https://nebiocalculator.neb.com/#!/ligation .


#There are three values to insert into the calculator.  
1. Insert these three values into the calculator.  
##Insert length in bp:  
:a. Length in bp:  
##Vector DNA length  
:b. Vector DNA length  
##Vector DNA mass: enter 50ng.  
:c. Vector DNA mass: enter 50 ng.
 
2. Use the (3:1) insert: vector ratio values.
 
3. Based on the amount of ng DNA resulting from the calculation, determine the appropriate volume of DNA to add to the ligation.
:a. '''Be sure to use the T4 DNA ligase (with 10x Ligase Buffer) protocol on NEB's website: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202'''
 
4. Thaw and resuspend ligase buffer at room temperature prior to performing the reaction.
 
:a. The exact volumes of vector and insert to add to the ligation reaction will depend on results from the NEBioCalculator.
1. Add 2 uL of 10X Ligase Buffer to a 0.6 mL tube.
 
2. Add the vector. How much? 50ng of DNA, so the volume will depend on the concentration of the vector.
 
3. Add three-times the molar amount of insert. This will be a variable amount; be sure to use the above calculator to find the volume needed.
 
4. Add 1 uL of T4 Ligase
 
5. Fill the remaining volume with molecular grade water (in 4 degree fridge) to 20 uL.


Go with the (3:1) insert:vector ratio values. Based on the amount of ng DNA resulting from the calculation, determine the appropriate volume of DNA to add to the ligation. BE SURE TO USE THE T4 DNA LIGASE (With 10X Ligase Buffer) protocol on NEB's Website: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202
6. Gently mix the reaction by gently pipetting up and down.
7. Allow the reaction to run for 10 minutes at room temperature.


Thaw and resuspend ligase buffer at room temperature prior to performing reaction.  
8. Heat inactivate at 65 degrees for 10 minutes.  


The exact volumes of vector and insert to add to the ligation reaction will depend on results from the NEBioCalculator.  
9. Either proceed to transformation, or store DNA in -20 degrees.
#Add 2uL of 10X Ligase Buffer to a 0.6mL tube.
#Add XX amount of insert
#Add XX amount of vector
#Add 1uL of T4 Ligase
#Fill remaining volume with molecular grade water (in 4 degree fridge) to 20uL.
#Gently mix the reaction by gently pipetting up and down.
#Allow reaction to run for 10 minutes at room temperature.
#Heat inactivate at 65 degrees for 10 minutes.
#Either proceed to transformation, or store DNA in -20 degrees.

Latest revision as of 10:00, 15 February 2024


Goal

  • Goal: To insert a fragment of DNA into a plasmid using restriction enzymes and inserts.

Pre-Protocol Questions

1. Are you familiar with the NEBioCalculator and NEB Ligation Protocol? If not, refer to the links below.

The protocols for digest and ligation are from New England Biolab's "NEB Cloner" tool: http://nebcloner.neb.com/#!/redigest. and NEBioCalculator:"https://nebiocalculator.neb.com/#!/ligation and NEB Ligation Protocol with T4 Ligase: https://www.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202.

Precautions

  • high DNA concentrations may result in an incomplete digest.

Restriction Enzymes in the MEE Lab (and Buffer to Use)

  • Acc651 (3.1 Buffer)
  • BamHI-HF (CutSmart Buffer)
  • BglII (3.1 Buffer)
  • BsaI-HF (CutSmart Buffer)
  • BsrGI-HF (CutSmart Buffer)
  • DraI (CutSmart Buffer)
  • EcoRI-HF (CutSmart Buffer)
  • EcoRV-HF (CutSmart Buffer)
  • FspI (CutSmart Buffer)
  • HindIII-HF (CutSmart Buffer)
  • KpnI-HF (CutSmart Buffer)
  • MluI-HF (CutSmart Buffer)
  • NcoI-HF (CutSmart Buffer)
  • NdeI (CutSmart Buffer)
  • NheI-HF (CutSmart Buffer)
  • PstI-HF (CutSmart Buffer)
  • PvuI-HF (CutSmart Buffer)
  • PvuII-HF (CutSmart Buffer)
  • SacI-HF (CutSmart Buffer)
  • SalI-HF (CutSmart Buffer)
  • SnaBI (CutSmart Buffer)
  • SphI-HF (CutSmart Buffer)
  • SspI-HF (CutSmart Buffer)
  • XhoI (CutSmart Buffer)

Digestion

1. Prior to digesting DNA, purify DNA amplified via PCR to remove primers and to change the buffer.

a. Do this with the Zymogen DNA Clean and Concentrator Kits above the microwave.

2. Beforehand, turn on 37 degrees water bath (this is for the incubation period) and dry bath at 80 degrees (if you are heat inactivating the restriction enzymes).

3. Answer these questions:

a. What volume of DNA you will need to add in order to digest 1 µg?
b. Are you digesting with multiple restriction enzymes that require different buffers? If so, please talk to Eric.
c. Are you stopping here overnight (or longer)? Then, after the digestion, heat inactivate the enzymes at 80 degrees for 20 minutes.

4.

For a 50 µl reaction:

DNA 1 µg
10X Buffer (CutSmart or 3.1, depending on the enzyme) 5 µl (1X)
Each enzyme 1.0 µl
Nuclease-free Water to 50 µl

OR

For a 10 µl reaction:

DNA 150 ng
10X Buffer (CutSmart or 3.1, depending on the enzyme) 1 µl (1X)
Each enzyme 0.2 µl
Nuclease-free Water to 10 µl

5. Incubate reactions in a 1.5mL microcentrifuge tube at 37 degrees for at least 1 hour.

6. If necessary, store this DNA in the -20 freezer or in the 4 degree refrigerator for later use.

7. Purify (with the DNA Clean and Concentrator Kit) both the insert and plasmid vector DNA separately to remove the restriction enzymes.

Ligation

Navigate to the NEBioCalculator: https://nebiocalculator.neb.com/#!/ligation .

1. Insert these three values into the calculator.

a. Length in bp:
b. Vector DNA length
c. Vector DNA mass: enter 50 ng.

2. Use the (3:1) insert: vector ratio values.

3. Based on the amount of ng DNA resulting from the calculation, determine the appropriate volume of DNA to add to the ligation.

a. Be sure to use the T4 DNA ligase (with 10x Ligase Buffer) protocol on NEB's website: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202

4. Thaw and resuspend ligase buffer at room temperature prior to performing the reaction.

a. The exact volumes of vector and insert to add to the ligation reaction will depend on results from the NEBioCalculator.

1. Add 2 uL of 10X Ligase Buffer to a 0.6 mL tube.

2. Add the vector. How much? 50ng of DNA, so the volume will depend on the concentration of the vector.

3. Add three-times the molar amount of insert. This will be a variable amount; be sure to use the above calculator to find the volume needed.

4. Add 1 uL of T4 Ligase

5. Fill the remaining volume with molecular grade water (in 4 degree fridge) to 20 uL.

6. Gently mix the reaction by gently pipetting up and down.

7. Allow the reaction to run for 10 minutes at room temperature.

8. Heat inactivate at 65 degrees for 10 minutes.

9. Either proceed to transformation, or store DNA in -20 degrees.

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