- PCR
- PCR Product Purification
- Agarose Gel Extraction
- Enzyme Digestion
- Ligation
- Transformation (& Competent Cell Production)
- DNA Preparation (Plasmid Isolation)
- Confirmation of Insertion (Enzyme Digestion & Colony PCR)
PCR
- (1) Prepare the mixture as follows in a PCR tube.
Composition Volume(µl) DNA Template (100ng) 2.5 10X buffer 10 dNTP mix, 10mM 2.0 Primer #1 (10uM) 2.5 Primer #2 (10uM) 2.5 Polymerase (1u/µl) 1.0 dH2O up to 100 Total 100 - (2) Pour the mixture into a PCR machine and react under the settings shown below.
PCR size < 10kbSteps # of Cycle Temperature (℃) Time (Mins) 1 1 95 5 95 1 2 30~35 Primer Tm-5* 1 72 PCR size 1/1kb 3 1 72 10 * This reaction is a general example. The optimal conditions must be determined through repeated tests.
(Annealing Temperature (*), Time, # of Cycle, etc.) - (3) Following PCR, store the product at 4℃.
- (4) Verify the product through electrophoresis in agarose gel. (Electrophoresis in 12% acrylamide gel can be used with products smaller than 100bp.)
PCR Product Purification
- (1) Mix the PCR product and buffer PBI (PCR purification buffer) in 1:5 ratio.
ex) 50µl PCR product with 250µl buffer PBI. - (2) Insert spin column in the provided 2ml collection tube.
- (3) In order to bind the DNA, pour the mixture (from step 1) in the column and centrifuge for 1 minute.
- (4) Discard the flow-through in the 2ml collection tube and reinsert the column in the tube.
- (5) Pour 750µl buffer PE in the column and centrifuge for 1 minute for washing.
- (6) Discard the flow-through collected in the 2ml collection tube. After reinserting the column into the tube, centrifuge again for another minute.
- (7) Transfer the column into a new 1.5ml tube.
- (8) For DNA elution, add an appropriate amount of buffer EB (10mM Tris-Cl, pH8.5) or D.W and let it sit for 1 minute.
(In order to maximize the amount of eluent, it is important to pour the buffer or the D.W to the center of the
membrane.) - (9) Following 1 minute centrifugation, remove the column
Agarose Gel Extraction
* Purifying 70bp - 10kb DNA from an agarose gel of TAE or TBE buffer.
- (1) Cut out the DNA band of interest from the agarose gel following electrophoresis
(Make sure to cut as precisely as possible). - (2) Weigh the gel.
- (3) Place the gel in the tube and add 3 times the amount of buffer QG as the weight of the gel.
(6 times the amount when using >2% gels) - (4) Incubate for 10 minutes at 50℃ until the gel completely dissolves.
* Vortexing every 2-3 minutes during incubation will quicken the dissolving process. After the gel has completely
dissolved, check to see if the mixture has turned yellow. If it is orange or purple, add 10µl 3M sodium acetate
(pH 5.0). - (5) Add the same amount of isopropanol as the weight of the gel. (Skip this step if the size of the DNA is 500bp-4kb)
- (6) Insert a spin column into the provided 2ml collection tube.
- (7) For DNA binding, add the prepared mixture from step 5 into the column and centrifuge for 1 minute.
- (8) Discard the flow-through collected in the 2ml collection tube and reinsert the column into the tube.
- (9) For washing, add 500µl buffer QG in the column and centrifuge for 1 minute.
- (10) Discard the flow-through collected in the 2ml collection tube. Reinsert the column and centrifuge for 1 minute.
- (11) Transfer the column into a new 1.5ml tube.
- (12) For DNA elution, pour an appropriate amount of buffer EB (10mM Tris-Cl, pH8.5) or D.W into the column and
let it sit for 1 minute. - (13) Following 1 minute centrifugation, remove the column from the tube.
Enzyme Digestion
Individual restriction enzymes have varying optimal temperature, reaction buffer, and thermal inactivation point. Therefore, it is important to test according to these individual conditions. Enzyme activity is measured in units. 1 enzyme unit is defined as the amount of enzyme required to digest 1 µg λ DNA in 50 µl reaction volume in 1 hour.
If the reaction conditions are not optimal during the digestion process, the amount of enzyme must be increased. However, because glycerol in enzymes can suppress enzyme activity, the amount of enzyme added should not exceed 10% of the entire reactant volume. Moreover, reaction times can be extended according to the type of enzyme and different characteristics it possesses.
The table below is a general example of a reaction. The conditions may vary according to the enzyme concentration and characteristics.
| Composition | Volume |
|---|---|
| DNA | 1µg |
| 10X reaction buffer | 2 µl |
| Enzyme (10unit/µl) | 0.1 µl |
| dH2O | Up to 20 µl |
| Total | 20 µl |
* 37℃ 1-2hrs incubation
Ligation
- (1) Prepare the below mixture in a 1.5ml tube.
Composition Volume(µl) Vector DNA (50-100ng) X Insert DNA X 10X Ligase Buffer 2 T4 DNA Ligase 1 dH2O Up to 20 Total 20 * Theoretically, the molar ratio of vectors and inserts should be 1:3, but this ratio can be adjusted depending on the preferences of the experimenter.
- (2) Mix the reactant using tapping motions.
- (3) Let it react for 4 hours at 16℃ or overnight at 4℃.
- (4) Conduct transformation in an appropriate E. coli strain.
Transformation
- (1) Take competent E.coli cell from -80℃ freezer and thaw in ice.
- (2) Carefully mix the prepared 10µl ligation reactant with the cell.
- (3) Incubate the reactant (from step 2) on ice for 30 minutes.
- (4) Put reactant into heat block (or water bath) at 42℃ for 1 minute.
- (5) Immediately transfer the reactant to an ice bath and leave for 5 minutes.
- (6) Add 500µl LB culture medium (10g tryptone, 5g yeast extract, 5g NaCl per liter) kept at 37℃ and incubate for
45 minutes at 37℃ (shake at 200rpm). - (7) Let the cells settle by centrifuging for 1 minute at 13,000rpm. Discard 500µl supernatant and suspend the cells
in the left over culture medium. - (8) Spread the cells onto an LB agar plates containing appropriate amount of antibiotics.
- (9) Incubate at 37℃ for 16 hours in an incubator.
Competent Cell Production
- (1) Inoculate the cell from the 5ml LB culture medium and shake incubate at 37℃ for 16 hours (shake at 200rpm).
- (2) In a flask that allows good aeration, add a new LB culture medium and inoculate the cell from step 1 at a 1:100 dilution ratio.
- (3) Incubate culture at 37℃ while shaking at 250RPM until OD600 = 0.4~0.6 (3-5 hours).
- (4) Transfer culture to pre-chilled sterile centrifuge tube, set on ice for 10 minutes.
- (5) Collect the cells after centrifuging for 10 minutes at 2,000rpm. Remove the supernatant.
- (6) Suspend the cells with pre-chilled 0.1 M CaCl2.
- (7) Place the suspended cells in ice for 1 hour.
- (8) Collect the cells after centrifuging for 10 minutes at 2,000rpm. Remove the supernatant.
- (9) Suspend the cells with pre-chilled 0.1 M CaCl2.
- (10) Place the suspended cells in ice for 1 hour.
- (11) Collect the cells after centrifuging for 10 minutes at 2,000rpm. Remove the supernatant.
- (12) Suspend the cells with pre-chilled 0.1 M CaCl2 and dispense 0.1ml into pre-chilled e-tubes.
- (13) Store the cells in -70℃ freezer.
-
0.1M CaCl2 (1L) CaCl2 14.7g Glycerol 150ml H2O up to 1L. * Sterilize by filtering
DNA Preparation (Plasmid Isolation)
- (1) Pick a single colony of cells from the plate and inoculate in 1-5ml LB culture medium.
Then, incubate in an incubator for 12-16 hours at 37℃.
* Incubation time can be shortened depending on the project. - (2) Harvest the cells by centrifugation for 3 minutes at 8,000rpm and removing the supernatant.
- (3) Add buffer 1 (Resuspension buffer) and vortex to completely suspend the cells. Transfer to a 1.5ml tube.
- (4) Add buffer 2 (Lysis buffer) and mix by inverting the tube 4-6 times.
*Important: Do NOT vortex at this stage as vortexing can cut the cell’s genomic DNA.) - (5) Add buffer 3 (Neutralization buffer) and immediately mix by inverting the tube 4-6 times.
- (6) Centrifuge for 10 minutes at 13,000 rpm.
- (7) Collect the supernatant and transfer to a spin column.
- (8) Following 30-60 second centrifugation, discard the flow-through collected in the 2ml collection tube and
reinsert the column in the tube. - (9) Add wash buffer into the column.
- (10) Following 30-60 second centrifugation, discard the flow-through collected in the 2ml collection tube and
reinsert the column in the tube. - (11) Centrifuge for additional 1-2 minutes and completely remove the remaining wash buffer.
- (12) Transfer the column to a new 1.5ml tube.
- (13) To elute DNA, add approximately 50µl of buffer EB (10mM Tris-Cl, pH8.5) or D.W and let it sit for 1 minute then
centrifuge for 1 minute. -
Buffer 1 (Resuspension buffer) 50mM Glucose 25mM Tris-Cl(pH8.0) 10mM EDTA Buffer 2 (Lysis buffer) 0.2N NaOH 1% SDS Buffer 3 (Neutralization buffer) 5M potassium acetate 60ml Glyacial acetic acid 11.5ml D.W up to 100ml
Confirmation of Insertion
- Enzyme Digestion
- * The separated plasmid DNA can be cut with restriction enzyme to check whether the insert DNA of interest has been correctly inserted.
-
Composition Volume DNA 1-2µg 10X reaction buffer 2.0 µl Enzyme 1 (10unit/µl) 0.1 µl Enzyme 2 (10unit/µl) 0.1 µ dH2O Up to 20 µl Up to 20 µl 1.0 Total 20 µl * 37℃ 1hr incubation
- * The separated plasmid DNA can be cut with restriction enzyme to check whether the insert DNA of interest has been correctly inserted.
- * The product can be checked via Agarose gel electrophoresis.
(Verify the size of vector and insert DNA used during cloning on the gel. If the vector and insert DNA is visible, there is a 99% chance that it is the DNA that you want.) - *The cloned product can be verified with the above method, but if you want to check for the direction of the insert (when using a one cut enzyme), or if you want to check for any mutations during PCR due to polymerase errors, it is necessary to use DNA sequencing to confirm the results.
- Colony PCR
- * This method is used for selecting the colony that has the inserts of interest prior to inoculating the colony for plasmid isolation.
- 1. Add 15.8µl dH2O in the PCR tube and pour the colony into the tube. Vortex carefully.
- 2. Add the reactants below into the tube.
-
Composition Volume (µl) 10X buffer 2.0 10mM dNTP 0.4 10pmol Primer 1 0.8 10pmol Primer 2 0.8 Taq DNA polymerase 0.2 * In general, Primer 1 uses forward primers within vectors and Primer 2 uses gene-specific reverse primers.
- 3. Pour the mixture (from step 2) in a PCR machine and react using the settings listed below.
PCR size < 10kbSteps # of Cycle Temperature (℃) Time (Mins) 1 1 95 5 95 0.5 2 25 Primer Tm-5* 0.5 72 1 (per 1kb) 3 1 72 10 -
- - The reaction above is a general example. Optimal conditions must be determined through repeated tests. (annealing temperature (*), time, # of cycles, etc.)
- - After running the PCR, store the product at 4℃.
- - Check the product through electrophoresis in agarose gel. (If under 100bp, can be verified via electrophoresis in 12% acrylamide gel.)