Subcloning TechniquesSubcloning Techniques

Created By: Sara Cormier

Hill Park Secondary School, The Co-Op & Science Departments and Mr. Watts

wish to thank

Dr. D. Tang

for allowing Sara Cormier to do a Co-Op placement in his lab (McMaster University/St. Joseph’s Hospital)

and

Dr. Jing Zhang,

a research fellow in Dr. Tang’s lab who both graciously gave of their time to supervise Sara’s work on a daily basis.

AcknowledgementAcknowledgement

What Does Subcloning Mean?What Does Subcloning Mean?

Subcloning is the transfer of a cloned DNA fragment from one vector to another.

What Is the Purpose?What Is the Purpose?

Protein expression is generally the purpose of subcloning.

The insert most likely will contain the gene that codes for the desired protein.

The effects of this protein on a cell can then be studied.

Steps Required To Steps Required To Successfully SubcloneSuccessfully Subclone

1. Obtain plasmid maps.

2. Select enzymes.

3. Perform 1st enzyme cut.

4. Precipitate.

5. Perform 2nd enzyme cut.

6. Gel purification.

7. Check plasmid concentration.

8. Ligation.

9. Transformation.

10. Screen.

Step 1: Obtain Plasmid MapsStep 1: Obtain Plasmid Maps

• Use plasmid map to select enzymes for ligation.

• Each enzyme provided on multiple-cloning site makes a single cut in the plasmid resulting in linear DNA.

• Plasmid maps also provide other important information such as which antibiotic resistance is present.

Best Case Scenario: Enzymes Best Case Scenario: Enzymes MatchMatch

If possible select the same 2 enzymes to cleave the vector and insert.

Enzymes that produce sticky ends are preferred as they make ligation easier.

Step 2: Perform 1Step 2: Perform 1stst Enzyme Enzyme CutCut

Purpose: to make the circular plasmid linear.

Cut vector and insert seperately.Select the appropriate buffer based on the

enzyme selected.If the enzymes are compatible (use the same

buffer) make both cuts at the same time.

Steps RequiredSteps Required

1. Determine how much plasmid should be used (2µg).

2. 10% of the total volume should be the buffer.

3. The enzyme should not be more than 10% of the total volume.

4. Add DNA H2O to make the final volume.5. Incubate at 37ºC overnight.

Step 3: Precipitate Vector and Step 3: Precipitate Vector and Insert Insert

Add 10% of the total volume sodium acetate (ph 5.2).

Add 2x the total volume 100% ethanol.Incubate on ice 20 min.Centrifuge 4ºC for 10 min on maximum

speed.

Step 4: Perform 2Step 4: Perform 2ndnd Enzyme Enzyme CutCut

Follow same basic procedure as the first cut.

1. Dissolve the pellet in water.

2. Add buffer and enzyme.

3. Incubate at 37ºC overnight.

Step 5: Gel PurificationStep 5: Gel Purification

Make DNA gel. Cut out the band

needed . Use a gel

purification kit to purify the DNA.

Use the molecular weight markers to determine which band is correct.

Step 6: Check Plasmid Step 6: Check Plasmid ConcentrationConcentration

Purpose: to know how much to use in ligation.

1. Make DNA gel.2. Run about 3 wells of both vector and

insert (different concentrations, all diluted).

3. Chose the band that is visible but not too dark.

Step 7: LigationStep 7: Ligation

Ligation – insert the insert into the plasmid to make a new plasmid (recombinant DNA).

1. Add the appropriate amount of vector and insert to a new tube (more insert than vector).

2. Add H2O (not always necessry).3. Use ligation kit and add:

- 2 µL buffer 2 (warm and vortexed).- 10 µL buffer 1 (warm and vortexed).- 1 µL buffer 3.

4. Incubate at room temperature for 15 min.

Step 8: Transformation:Step 8: Transformation: Transformation - introduction of foreign DNA, in

this case a plasmid into a bacterial cell.

Steps RequiredSteps Required

1. Add total volume of ligated plasmid to a competent cell.

2. Incubate on ice for 30 min.3. Heat shock (42ºC for 90 sec.).4. Incubate on ice for 2 min.5. Add to 0.8mL LB medium and shake at 37ºC for

1 hour.6. Centrifuge and spread on ampicillin plate (if this

is the antibiotic being used).7. Incubate overnight at 37ºC.

Step 9: ScreenStep 9: Screen Purpose: to determine if the

ligation was successful. Select 2 enzymes (one from

vector and one from insert). Based on the bands

appearing in the gel you can determine if ligation was successful.

The insert orientation must be checked if the same enzyme was used or blunt ligation was used.

Example:Example:

In the above example 6 samples were run. The 3rd and 4th samples were successful.

1 2 3 4 5 6Sample

Steps RequiredSteps Required

1. Pick up colonies (number varies) from plate and place each in 5mL LB medium and (3 µL antibiotic).

2. Shake overnight at 37°C.3. Centrifuge 1mL from each tube for 5 min.

speed 2000rpm at room temperature.4. Make enzyme cuts together.5. Run gel to check if ligation successful.

What Happens When the What Happens When the Enzymes Don’t Match?Enzymes Don’t Match?

Subcloning becomes more difficult and less successful when it is not possible to cleave the insert and vector with the same 2 enzymes.

Steps To Successfully Steps To Successfully Subclone When Only ONE Subclone When Only ONE

Enzyme MatchesEnzyme Matches1. Obtain plasmid maps.2. Select enzymes.3. Perform 1st enzyme

cut (enzyme that does NOT match).

4. Blunt vector & insert.6. Perform 2nd enzyme

cut (enzyme that matches).

7. Gel purification.

8. Check plasmid concentration.

9. Ligation.

10. Transformation.

11. Screen.

General ExampleGeneral Example

Blunting: Get Rid of the Blunting: Get Rid of the OverhangsOverhangs

In order to allow for ligation the overhangs must be removed by a process known as blunting.

This step may be eliminated if possible to cut with an enzyme that produces blunt ends.

Any blunt ends can ligate together.

Steps RequiredSteps Required

1. In small PCR tubes add:

- 34 µL H2O

- 5 µL Pfu buffer (10x buffer) (warm)

- 10 µL 1mm DNTP (warm)

- 1 µL Pfu enzyme

Total volume: 50 L• Centrifuge to get rid of any bubbles.• In PCR machine heat at 72ºC for 30 min.

Steps To Successfully Steps To Successfully Subclone When NO Enzymes Subclone When NO Enzymes

MatchMatch1. Obtain plasmid maps.2. Select enzyme.3. Perform 1st enzyme

cuts.4. Perform 2nd cut on

insert.5. Blunt vector & insert.6. Dephosphorylate

(vector only).

7. Gel purification.

8. Check plasmid concentration.

9. Ligation.

10. Transformation.

11. Screen.

General Example:General Example:

DephosphorylationDephosphorylation

Dephosphorylation – to remove the phospate group from the 5’ ends on the vector.

Purpose: insures that the vector does not self-ligate.

Steps RequiredSteps Required

1. Precipitate vector2. In separate tube mix: - 9 µL H2O - 1 µL AP bufferThrow out 1 µL from tube and

replace with 1 µL phosphatase.

3. To precipitated vector add:

- 44 µL H20 - 5 µL AP buffer - 1 µL mix (from above)

4. In PCR machine set: - 15 min. 37°C - 45 min. 55°C - 15 min. 74°C5. After the first 15 min. add

another 1 µL of the mix.6. After 1 hour add 1 µL 0.5M

EDTA7. After completed put on ice.

My ProjectsMy Projects

pcDNA 3.0/BCL-2 (sticky end ligation)PBind/P58 (blunt end ligation)pcDNA 3.1/hygro/BCL-2 (sticky end

ligation)

pcDNA 3.0/BCL-2pcDNA 3.0/BCL-2

Checking Concentration (to know how much to ligate):

BCL-2 pcDNA 3.0

pcDNA 3.0/BCL-2pcDNA 3.0/BCL-2Screening:

All 8 samples were successful.

pcDNA 3.1/hyrgo/BCL-2pcDNA 3.1/hyrgo/BCL-2

The pupose of inserting BCL-2 into this vector is that it contains hygromycin. This selection marker was required during transfection.

THANK-YOUTHANK-YOU