Cell lines available for cloning

1. Human Adrenal Cortical Cells 
2. Human Type II Alveolar Epithelial Cells 
3. Human Pulmonary Alveolar Epithelial Cells 
4. Human Oral Epithelial Cells 
5. Pig Muscle Satellite Cells 
6. Human Retinal Pigment Epithelial Cells 
7. MKN-1 
8. huH-1 
9. NUGC-4 
10. MKN-7 
11. MDA-MB-468 
12. M-07E 
13. COV362 
14. BV-2 
15. BJAB 

Primary Cells

1. - GFP or RFP Expressing Cells
2. - Human Primary Cells
3. - Motion System Primary Cells
4. - Nervous System Primary Cells
5. - Endocrine System Primary Cells
6. - Digestive System Primary Cells
7. - Blood System Primary Cells
8. - Urinary System Primary Cells
9. - Reproductive System Primary Cells
10. - Immune System Primary Cells
11. - Respiratory System Primary Cells
12. - PBMCs
13. - Others
14. - Animal Primary Cells
15. - Mouse Primary Cells
16. - Rat Primary Cells
17. - Bovine Primary Cells
18. - Monkey Primary Cells
19. - Rabbit Primary Cells
20. - Porcine Primary Cells
21. - Other Animal Primary Cells
22. - Subcellular Fractions

Tumor Cell Lines

1. - Human Tumor Cell Lines
2. - Animal Tumor Cell Lines

MicroRNA Agomir/Antagomir

1. - Human MicroRNA Agomir/Antagomir
2. - Mouse MicroRNA Agomir/Antagomir
3. - Rat MicroRNA Agomir/Antagomir
4. - Other MicroRNA Agomir/Antagomir

Stem Cells

1. - Embryonic Stem (ES) Cells
2. - Adult Stem Cells
3. - Cancer Stem Cells
4. - Mesenchymal Stem Cells (MSC)
5. - Induced Pluripotent Stem Cells (iPSCs)

Immortalized Cell Lines

Transfected Stable Cell Lines

1. - Knockout Stable Cell Lines hot products
2. - Knockin Stable Cell Lines
3. - Knockdown Stable Cell Lines hot products
4. - Overexpression Stable Cell Lines hot products
5. - Reporter Stable Cell Lines
6. - Knockout Cell Lysate
7. - In-Stock Stable Cell Lines

Nucleic Acid Kits

• 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 < 10kb

  Steps	# 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 < 10kb

  Steps	# 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

• 1. - The reaction above is a general example. Optimal conditions must be determined through repeated tests. (annealing temperature (*), time, # of cycles, etc.)
  2. - After running the PCR, store the product at 4℃.
  3. - Check the product through electrophoresis in agarose gel. (If under 100bp, can be verified via electrophoresis in 12% acrylamide gel.)