2AcknowledgementHill Park Secondary School, The Co-Op & Science Departments and Mr. Wattswish to thankDr. D. Tangfor allowing Sara Cormier to do a Co-Op placement in his lab (McMaster University/St. Joseph’s Hospital)andDr. 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.
3What Does Subcloning Mean? Subcloning is the transfer of a cloned DNA fragment from one vector to another.
4What 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.
6Step 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.
7Best Case Scenario: Enzymes Match 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.
8Step 2: Perform 1st Enzyme Cut 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.
9Steps Required Determine how much plasmid should be used (2µg). 10% of the total volume should be the buffer.The enzyme should not be more than 10% of the total volume.Add DNA H2O to make the final volume.Incubate at 37ºC overnight.2ug becase too much not all cut, just enough so enough left at end, some lost during steps
10Step 3: Precipitate Vector and 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.
11Step 4: Perform 2nd Enzyme Cut Follow same basic procedure as the first cut.Dissolve the pellet in water.Add buffer and enzyme.Incubate at 37ºC overnight.
12Step 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.
13Step 6: Check Plasmid Concentration Purpose: to know how much to use in ligation.Make DNA gel.Run about 3 wells of both vector and insert (different concentrations, all diluted).Chose the band that is visible but not too dark.
14Step 7: LigationLigation – insert the insert into the plasmid to make a new plasmid (recombinant DNA).Add the appropriate amount of vector and insert to a new tube (more insert than vector).Add H2O (not always necessry).Use ligation kit and add:- 2 µL buffer 2 (warm and vortexed).- 10 µL buffer 1 (warm and vortexed).- 1 µL buffer 3.Incubate at room temperature for 15 min.
15Step 8: Transformation: Transformation - introduction of foreign DNA, in this case a plasmid into a bacterial cell.
16Steps RequiredAdd total volume of ligated plasmid to a competent cell.Incubate on ice for 30 min.Heat shock (42ºC for 90 sec.).Incubate on ice for 2 min.Add to 0.8mL LB medium and shake at 37ºC for 1 hour.Centrifuge and spread on ampicillin plate (if this is the antibiotic being used).Incubate overnight at 37ºC.
17Step 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.
18Example:#2 uncut #5 no insertSampleIn the above example 6 samples were run. The 3rd and 4th samples were successful.
19Steps RequiredPick up colonies (number varies) from plate and place each in 5mL LB medium and (3 µL antibiotic).Shake overnight at 37°C.Centrifuge 1mL from each tube for 5 min. speed 2000rpm at room temperature.Make enzyme cuts together.Run gel to check if ligation successful.
20What Happens When the 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.
21Steps To Successfully Subclone When Only ONE Enzyme Matches 1. 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.
23Blunting: Get Rid of the Overhangs 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.
24Steps Required In small PCR tubes add: - 34 µL H2O - 5 µL Pfu buffer (10x buffer) (warm)- 10 µL 1mm DNTP (warm)- 1 µL Pfu enzymeTotal volume: 50 LCentrifuge to get rid of any bubbles.In PCR machine heat at 72ºC for 30 min.
25Steps To Successfully Subclone When NO Enzymes Match 1. 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.
27DephosphorylationDephosphorylation – to remove the phospate group from the 5’ ends on the vector.Purpose: insures that the vector does not self-ligate.
28Steps Required Precipitate vector In separate tube mix: - 9 µL H2O - 1 µL AP bufferThrow out 1 µL from tube and replace with 1 µL phosphatase.To precipitated vector add:- 44 µL H20- 5 µL AP buffer- 1 µL mix (from above)In PCR machine set:- 15 min. 37°C- 45 min. 55°C- 15 min. 74°CAfter the first 15 min. add another 1 µL of the mix.After 1 hour add 1 µL 0.5M EDTAAfter completed put on ice.
29My Projects pcDNA 3.0/BCL-2 (sticky end ligation) PBind/P58 (blunt end ligation)pcDNA 3.1/hygro/BCL-2 (sticky end ligation)
30pcDNA 3.0/BCL-2 Checking Concentration (to know how much to ligate): BCL pcDNA 3.0
31pcDNA 3.0/BCL-2Screening:All 8 samples were successful.
32pcDNA 3.1/hyrgo/BCL-2The pupose of inserting BCL-2 into this vector is that it contains hygromycin. This selection marker was required during transfection.