DNA Electrophoresis on Agarose Gels

Making agarose gel stock (100 mL):

1. Weigh agarose powder on a balance. The amount of agarose needed depends on the concentration of the gel that will be used. The higher the concentration, the resolving ability is also higher.
2. For agarose gel 1% = 1 gram of agarose powder

1.5% = 1.5 grams of agarose powder

2% = 2 grams of agarose powder

3. Mix agarose powder with 1X TAE buffer in a heat-proof bottle. Dissolve it by heating in microwave oven on medium high for several minutes (use no lid or loosen lid while heating).
4. Occasionally during heating, shake the bottle to help the agarose dissolve.
5. Make sure the agarose dissolve completely (the mixture will be absolutely clear and show no traces of undissolved powder). Be careful not to overheat as the agarose mixture will overflow.
6. The agarose mixture is ready to be used directly or stored.

Electrophoresis preparation:

1. Prepare the comb and plate for pouring melted agarose. Use an appropriate comb depending on the number of samples that will be run.
2. Wait until the liquid agarose mixture cools down to around 60 ^oC (cool enough to not scorch your hand but not too cool that it partly solidifies). DO NOT POUR BOILING AGAROSE MIXTURE ON PLATE!
3. Pour agarose mixture on plate; the volume will depend on the volume of samples to be put in wells. For a reference, 30 mL of agarose mixture poured on plate with a standard 8-well comb will hold a maximum of 20µL of sample in each well.
4. Leave the agarose gel to set (around 10—15 minutes).
5. When the gel is set, take out the comb and put the plate in electrophoresis tank.
6. Pour enough 1X TAE buffer to cover the gel and fill the tank (approximately 250 mL buffer).

Loading samples and running electrophoresis:

1. Take out the samples, loading dye (2X), and DNA marker/ladder from fridge/freezer. Thaw completely.

2. Mix samples with loading dye on a parafilm sheet. As a guide, 2 µL of loading dye is enough to hold down 4—5 µL of sample. For larger amounts of samples, increase the amount of loading dye accordingly. Pipette up and down using a micropipette to mix the sample and loading dye completely.

3. Carefully pipette the sample/dye mixture into each well. After all samples are loaded into wells, load 4 µL of DNA ladder into one well. Note: the DNA ladder being used here is ready-to-use (already mixed with loading dye). If not, mix it with appropriate amount of loading dye first before pipetting it into the well.

4. Assemble the lid on tank and connect it to a power source. Make sure the side of gel that has wells is placed on the side of tank that is connected to the cathode (negative) of power source, as DNA is negatively charged and will travel to positively charged side (anode).

5. Set the current to maximum and set the voltage to 60-80 volts.

6. Run the electrophoresis for around 1—2 hours or until the loading dye reaches ¾ of gel (higher concentration of gel and lower voltage means longer running time). Do not let it run for too long or the DNA will be lost in the buffer.

Visualising DNA fragments:

1. When electrophoresis is finished, turn off power and open the tank. Take out the gel and put it on a container.
2. Pour EtBr (ethidium bromide) solution (30 µL in 500 mL of water) over the gel until it is completely soaked.
3. Soak the gel for 10—15 minutes. If the ethidium bromide solution is old or has been re-used many times, leave the gel soaked for longer.
4. Afterwards, pour back EtBr solution back in its bottle and wash the gel with tap water for 2—3 minutes.
5. Visualise DNA by viewing the gel under UV light.
6. If the DNA fragment is too faint, soak the gel again in EtBr solution. If the gel is too bright (soaked too long in EtBr), increase washing time to 5—6 minutes.
7. Photograph the gel with a manual polaroid camera or with GelDoc System.

Competent Cells

CaCl₂ METHOD

(All activities are done in aseptically in laminar flow cabinet, except for centrifuging).

1. Prepare overnight culture – pick a colony of E. coli on agar plate and grow in 3 mL LB broth. Incubate at 37 ^oC overnight.
2. Use 1 mL overnight culture to inoculate 100 mL LB broth in 500 mL erlenmeyer flask.
3. Incubate at 37 ^oC with shaking (200 rpm).
4. Grow to OD 600 = 0.4—0.5 (optimal for DH5α). Usually takes about 3 hours.
5. Make 2 x 50 mL aliquots in oakridge tubes, centrifuge at 500 rpm for 15 minutes. Discard supernatant.
6. Resuspend pellet in 10 mL ice cold CaCl₂ solution.
7. Centrifuge at 5000 rpm for 15 minutes, discard supernatant.
8. Resuspend each pellet in 1 mL ice cold CaCl₂ solution.
9. Combine and make 200 µL aliquots in sterile 1.5 mL microcentrifuge tubes.
10. Snap freeze on dry ice/ETOH.
11. Store at -70 ^oC, labelled CC.

For transformation (heat-shock method):

1. Mix 100—200 µL of CC with 1 µL plasmid DNA (or 10 µL ligation mixture) in a sterile 1.5 mL microcentrifuge tube. Incubate on ice (or put in freezer) for 30 minutes.
2. Heat shock treatment: put tube in dry bath or water bath at 42 ^oC for 2 minutes.
3. Immediately put on ice for 10 minutes.
4. Cell/DNA mixture is spread on agar plate (plus ampicillin or other antibiotic). Incubate at 37 ^oC overnight.

TSS METHOD (1)

1. Inoculate 3 mL of LB broth with an E. coli colony from agar plate. Incubate at 37 ^oC overnight, with shaking.
2. Dilute 1:100 fresh overnight culture of bacteria into pre warmed LB broth and incubate cells with shaking (225 rpm) to an OD 600 of 0.3—0.4 (approximately 3 hours).
3. Add an equal volume of ice-cold 2X TSS and mix gently. [TSS is LB broth with 10% PEG (MW 3350-8000), 5% DMSO, and 20-50 mM Mg ²⁺ (MgSO₄ or MgCl₂) at a final pH of 6.5].
4. For long term storage, cells are frozen immediately in a dry ice/ethanol bath and stored at -70 ^oC.

TSS METHOD (2)

1. Inoculate 50 mL LB broth with 0.5 mL fresh overnight culture and incubate at 37 ^oC with shaking until OD 600 reaches 0.3-0.4.
2. Spin to pellet cells after mixing (5000 rpm, 3 minutes) and add 2 mL of 1 X TSS.

For transformation (1):

1. A 0.1 mL aliquot of competent cells pipetted into a cold polypropylene tube containing 1 µL (100 pg) of plasmid DNA or ligation mixture, and cell/DNA suspension is mixed gently. [When frozen cells are used, cells are thawed slowly on ice and used immediately].
2. The cell/DNA mixture is incubated for 5—60 minutes at 4 ^oC.
3. A 0.9 mL aliquot of TSS (or LB broth) plus 20 mM glucose is added, and cells are incubated at 37 ^oC with shaking (225 rpm) for 1 hour to allow expression of the antibiotic resistance gene.
4. Transformants are selected with standard methods.

another Molecular Biology Reagents

PMSF 100mM

Dissolve 17.4 mg/mL in isopropanol

Store at -20C

30% Acrylamide/bis

72.5 mL Acrylamide 40%

1 g bis-acrylamide

Dissolve in water until 100 mL

TBS

10 mM Tris-HCl pH 7.5

15 mM NaCl

Sterilize by autoclaving

IPTG 100 mM

For 20 mL, dissolve 476.6 mg IPTG in 20 mL water

TTBS

TBS with 0.1% of Triton X-100

1 Kb dna ladder marker

Stock = 500 ug/uL

Dilute to 125 ng/uL

For work : 100 uL marker + 100 uL 1XTE + 200 uL 2XLB

100 bp dna ladder marker

Stock = 500 ug/uL

Dilute to 125 ng/uL

For work : 100 uL marker + 100 uL 1XTE + 200 uL 2XLB

1 Kb dna ladder marker

Stock = 250 ug (1 ug/uL)

Dilute to 125 ng/uL (1:8)

For work : 50 uL marker + 150 uL water + 200 uL 2XLB

Tris-Glycine Electroforesis Buffer 5X Stock

15.1 g tris base

94 g Glycine

900 mL water

50 mL SDS 10%

Addjust volume with water until 1000 mL

2XSB

100 mM DTT (Stock 1M)

2% SDS

80 mM Tris-HCl pH 6.8

0.006% w/v bromophenol blue

15% Glycerol

Agarose TOP (100 mL)

LB powder for 100 mL

100 mg MgCl2

700 mg Agarose

Sterilize with autoclaving, store in refrigerator

Inclusion Body wash Solution 1 (Cold)

2 M urea

20 mM Tris

0.5 M NaCl

2% Triton X-100

Adjust to pH 8.00

Inclusion Body Wash Solution 2 (Cold)

20 mM Tris

0.5 M NaCl

2% Triton X-100

Adjust to pH 8.00

Solubilisation Buffer

20 mM Tris

0.5 M NaCl

5 mM Imidazole

6 M Guanidine HCl

1 mM Beta-mercaphtoethanol (BME)

Adjust to pH 8.00

(Concentration of BME might have to be optimized)

Equilibration buffer

300 mL ethanol 95%

95 mL Glycerol 50%

555 mL water

Molecular Biology Reagents

Luria Broth LB

(prepare with dd H2O, sterlilized by autoclaving)

1% w/v bacto-tryptone

0.5% w/v yeast extract

1% w/v NaCl

pH adjusted to 7.0 using NaOH 0,6 N

Solution 1 ( Cell Resuspension Solution)

50 mM Glucose (4.5 mL; stock 20%)

25 mM Tris pH 8 (2.5 mL; stock 1 M)

10 mM EDTA (10 mL; stock 0.1 M)

Solution 2 ( Cell Lysis Solution, Fresh)

2 M NaOH 1 mL

10 % SDS 1 mL

Dd H2O 8 mL

Solution 3 ( Neutralisation Solution)

5 M potassium acetate 60 mL

Glacial cetic acid 11.5 mL

H2O 28.5 mL

2X TSS (Competent cells media, TSS methods)

TSS=LB + 10% PEG + 5% DMSO + 20-50 mM MgCl2

pH=6.5

Glycerol-CaCl2 Solution (competent cells media, CaCl2 methods)

22.5 mL 0.4 M CaCl2

28.1 mL 80% Glycerol

99.4 mL H2O

TAE 50X/L

242 g Tris Base

57.1 mL Glacial acetic acid

100 mL 0.5 M EDTA pH 8

TBE 5X/L

54 g Tris Base

27.5 g Boric Acid

20 mL 0.5 M EDTA pH 8

EtBr

Stock Solution 10 mg/mL ( 1 tablet / 1 mL H2O)

Working Solution 0.5 ug/mL (250 mL H2O)

Double Detergent Lysis Bufer (100 mL)

50 mM Tris-HCl pH 8

150 mM NaCl

0.1% SDS

1% IGEPAL

SDS-PAGE Gel

12% resolving gel (1 gel)

3.4 mL H2O

2.5 mL 1.5M Tris-HCl pH 8.

50 uL SDS 20%

4 mL Acrylamida/bis

50 uL APS 10%

5 uL TEMED

12% stacking gel (2 gel)

3.075 mL H2O

1.25 mL Tris-HCl pH 6.8

25 uL SDS 20%

0.67 mL Acrylamida/bis

25 uL APS 10%

5 uL TEMED

8% Resolving gel (2 gel)

9.3 mL H2O

5.0 mL Tris-HCl pH 8.8

0.1 mL SDS 20%

5.3 mL Acrylamida/bis

0.2 mL APS 10%

0.012 mL TEMED

8% Stacking gel = 12% Stacking gel

Low Toxicity Staining Solution

0.25 g coomassie blue R250

100 mL ethanol

100 mL water

Stir 1 hour

+ 25 mL Acetic Acid

+water until 250 mL

Stored at room temperature (Alumunium foiled bottle)

Destaining Solution

400 mL ethanol

100 mL acetic acid

500 mL water

Stored at room temperature