UCLA

UNDERGRADUATE RESEARCH TEAM

TRAINING | PART 2
 P U R P O S E

The last presentation gave a general overview of the cloning process in

terms of the purpose of each step. It went in depth into PCR and primer
design. This presentation is meant to elaborate on the rest of the steps (e.g.
mechanisms, protocols, etc.)!
M A I N T O P I C S

GEL ELECTROPHORESIS

GEL EXTRACTION

RESTRICTION DIGEST

LIGATION

TRANSFORMATION
 M E C H A N I S M

Separation of DNA, RNA, or proteins according to molecular size.

Molecules are pushed by an electric field through a porous gel. Smaller
molecules move faster.

DNA and RNA are negatively charged. Proteins must be treated with SDS
to coat them with a negative charge.

After gel electrophoresis, bands representing molecules of different sizes

can be detected.
 P R O T O C O L : A G A R O S E

1. Make a 1% agarose gel.

a. Can increase the percentage (don’t go higher than 2%) if you need better
separation of bands.
b. Volume depends on how much well volume you need. Usually 100 - 150mL
is sufficient.
c. Add SybrSafe for staining.
2. Place well comb while gel is still liquid. Wait 20 min.

TIP: if you forgot about your gel in the flask and it solidified, don’t throw it out! Just microwave it again.
 P R O T O C O L : A G A R O S E

3. Add dye to your sample molecules (if necessary) and ladder. This lets the
sample sink to the bottom of the well.
4. Load each sample into a separate well, leaving one well for the ladder.
5. Make sure the gel box is plugged in correctly!
6. Switch current on and wait until samples have run a sufficient distance
(~60% down) before imaging.
 L A D D E R

There are many different sized ladders, depending on how large your
fragments are (DON’T USE THE WRONG LADDER).
e. g. 100bp, 50bp, 1kb ladder

These contain fragments with known sizes to allow for comparison with your
samples.
 F A C T O R S A F F E C T I N G R E S O L U T I O N

1. V O L T A G E :
a. On average, run at 5V per cm (from anode to cathode). Usually ~100 - 120V.
b. Lower voltage will decrease mobility but lead to higher resolution.

2. G E L C O N C E N T R A T I O N :
a. Higher gel percentage results in thinner bands.

Balance efficiency with resolution!

 S O L U B I L I Z E G E L

1. Record weight of a 1.5 mL centrifuge tube.

2. Cut your band of interest using a razor blade and a blue light imager.
a. Try to cut as close to the band as possible
3. Record weight of tube + gel
4. Add an appropriate amount of ADB (3:1 vol ratio of ADB : agarose)
a. 1mg agarose is 100uL of agarose
5. Dissolve completely (should not see swirling if tube is inverted).
6. See kit for further instructions.
 B I N D

DNA must bind to the column (made from silica). Centrifuged to pass
solution through.
 W A S H

Flow-through from binding step discarded. Wash buffer added (high

ethanol content) to remove any remaining salts and impurities.

Dry spin to remove the ethanol and ensure a clean eluent.

 E L U T E

EB buffer : buffers pH to stabilize DNA

TE buffer: added EDTA can inhibit DNases; inhibits enzymatic reactions

Water: no salts → good for transformations

Elution can be maximized by allowing the buffer to sit in the membrane

for a while to completely rehydrate the DNA.
 P R O T O C O L

CHECK THE PROTOCOL THAT COMES WITH EACH KIT! You may have
to modify these protocols depending on your yield (adding a dry spin step
and an incubation at RT step after adding elution buffer usually helps).

*Don’t forget to add ethanol to wash buffers (and mark that you did so!)
**Don’t elute in less than what they recommend. Each column has a
specific binding capacity and you want as much of your DNA as possible.
***If using DNA CnC-5, make sure you pay attention to how much binding
buffer to add.
R E S T R I C T I O N D I G E S T
 R E S T R I C T I O N E N Z Y M E S

Endonucleases that cleave DNA at specific recognition sequences. Type II

enzymes cut DNA close to or within their recognition site.

Can create sticky ends and blunt ends (less efficient for ligation).
U S E I N C L O N I N G
 U S E I N C L O N I N G

Double digests (cutting with two different restriction enzymes) may be

used for directional cloning.
 P R O T O C O L

COMPONENT AMOUNT

Restriction Enzyme 10 units generally sufficient (1uL)

10X NEBuffer 5 uL (1X)

Total Rxn Volume 50 uL

Incubation Time 1 hour

Incubation Temperature Enzyme dependent

DNA 1 ug
 C H O O S I N G T H E B U F F E R

Buffer selection is extremely important for efficient reactions. Use the

NEB double digest finder and NEB cloner in order to choose the right
buffer and protocol based on your restriction enzyme(s).

*some restriction enzymes are not compatible to be used together.

**HiFi versions of enzymes need different buffers than regular enzymes.
 H E A T I N A C T I V A T I O N

Halts a digestion reaction by inactivating the enzymes with heat. Check

NEB Heat Inactivation chart to find out required temperature and time
duration.

*Some enzymes cannot be heat inactivated (like BamHI). You must purify
these digestions to get rid of the enzyme.
 S T A R A C T I V I T Y

Sometimes restriction enzymes can cleave sequences similar but not

identical to their recognition site.

Possible reasons:
● high concentrations of enzyme or DNA
● non-optimal buffer
● prolonged reaction time
● presence of organic salts
 M E T H O D N O T E

There are many molecular cloning techniques that do not require digestion!
For example: TA cloning and Gibson assembly.

It’s up to you to decide which cloning strategy best fits your project.
L I G A T I O N
 M E C H A N I S M

DNA ligase uses ATP to catalyze the formation of a bond between the 5’
phosphate group of one DNA strand to the 3’ OH group of the other.
 C O M P O N E N T S

COMPONENT 20 uL Reaction

T4 DNA Ligase Buffer (10X) 2 uL

Vector DNA 50 ng

Insert DNA Variable

Nuclease-free water to 20 uL

T4 DNA Ligase 1 uL

*vector to insert molar ratio of 1:3 needed (use NEB ligation calculator)
**add DNA ligase last
***completely thaw T4 DNA Ligase Buffer before use
 P R O T O C O L

1. Gently mix reaction by pipetting up and down.

2. For sticky ends, incubate at 16 C overnight (or RT for 10 minutes but
overnight is recommended).
3. For blunt ends or single bp overhangs, incubate at 16 C overnight (or RT
for 2 hours).
4. Heat inactivate at 65 C for 10 min.
5. Chill on ice and transform 2 uL of the reaction into 30 uL competent
cells.
T R A N S F O R M A T I O N
 E L E C T R O P O R A T I O N

Physical transfection method. Electric pulse creates temporary pores in

cell membranes through which DNA can enter.

Easy and rapid, but causes substantial cell death. Needs more cells and
immediate rescue with SOC (1 minute in delay can cause a 3 fold
reduction in transformation efficiency).

If you hear a POP, you had salt in your solution!

 E L E C T R O P O R A T I O N

For use with electrocompetent cells only.

1. Pre-chill electroporation cuvettes (1mm) on ice.
2. Thaw electrocompetent cells on ice (IMMEDIATELY TRANSFER
FROM FREEZER TO ICE).
a. Aliquot 30uL to small PCR tube.
3. Return cells to freezer.
4. Add 2uL of DNA solution (NO SALT -- make sure you eluted in water)
to cells. Flick gently to mix.
5. Transfer all of the mixture to the cuvette (NO BUBBLES).
 E L E C T R O P O R A T I O N

6. Have 970 uL of SOC and 1.5mL tube ready!

7. Insert cuvette (with bump facing inside toward pulser) until you meet
some resistance.
8. Pulse (1.8kV w/ 1mm cuvettes or 2.5 kV w/ 2mm cuvettes)! Arc time
should be approximately 4 - 5 msec.
9. IMMEDIATELY add SOC to cuvette. Pipette up, down, up, and then
transfer to the 1.5mL tube.
10. Shake at 800 rpm at 37 C for 1 hour.
11. Pre-warm selective plates.
12. Do serial dilutions of cells onto plates and incubate at 37 C overnight.
 O T H E R M E T H O D S

Heat shock and chemical transfection methods can also be used. Look
online for protocols that are tailored to your specific cell (if using
commercial cells).

You may also have to make your own competent cells. Although possible,
we’ve never actually gotten this to work.
Q U E S T I O N S ?