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Q5® High-Fidelity DNA Polymerase PCR

1. Calculate the annealing temperature:

https://tmcalculator.neb.com/#!/main
Hint: From benchling take only the blue one to calculate annealing

temperature.
2. Set up the reaction on ice.
Use 15.75 µl of water for a 25µl reaction if an enhancer is not used
(usually not used).

Verify PCR successfulness through 1% agarose gel.

Agarose Gel (for 1% gel)

Note: For Gel Extraction, use 2% gel and load ~18µl samples (from step 1:
PCR + loading dye). For just analysis, 1% gel is OK.

Making Gel

1. Add 0.5 g Agarose to 50 ml 1X TAE (Tris-Acetate-EDTA) in a clonical

flask.
2. Microwave the solution until the agarose is dissolved in the TAE.
3. Let it cool, and add 5µl Apex Gel Stain (mix) when it is cooled down to
~55-50˚C.
4. Pour into a gel tray with a well-comb and let it solidify.
5. If needed, store it at 4˚C in a zip lock bag with a little bit of 1X TAE.
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Running Gel

1. Get a new 1.5ml centrifuge tube and add 5µl PCR product and 1µl 6x
purple loading dye.
2. Add 1X TAE until the gel is covered with it.
3. Load gels. Usually, 4µl Ladder and 5 µl sample (from step 1: PCR +
loading dye).
4. Note the order of samples/labels in the lab notebook.
5. Run it for about 20~30 min at 130 Volts. (from black/cathode/negative-
electrode to red/anode/positive-electrode).

1% Gel 2% Gel
Agarose 0.5 gram 1 gram
1X TAE 50 ml 50ml

Imaging Agarose Gel

1. For DNA, use Blue Sample Tray; for the SDS page and Western Blot, use
Blot/UV/Stain-Free Sample Tray (Purple/Green).
2. User Manual for Imager:
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10000062126.p
df
3. Load the Blue Sample Tray(or appropriate) in the imager.
4. Login
5. Under application, select “Nucleic Acid Gels> GelGreen Gel. (select the
all-blue one).”
6. Put Gel in the tray, close the door, and click on Camera > to take
images.
7. Go to the gallery to view the images.
8. Best Practice: Rename the images with Date Ordered Labels and
Purpose and your initials. If not, at least provide your date and initials.
9. You can transfer the image to the Pendrive/Flashdrive/Thumbdrive/USB.
The image can be analyzed using “Image Lab” software.
10. Clean the Blue Sample Tray.
11. Logout.

Gel Extraction

1. Calculate the mass of the centrifuge tube.

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2. While cutting gel, protect yourself and the Gel from overexposing to UV
light. The DNA can mutate in UV light. Cut as precise the gel as
possible. (Cut as small fragments as possible without losing a band. It’s
good to go for a larger fragment than losing a band.)
3. Follow the protocol,
(Zymoclean™ Gel DNA Recovery Kit)
https://files.zymoresearch.com/protocols/_d4001t_d4001_d4002_d4007
_d4008_zymoclean_gel_dna_recovery_kit.pdf?
_gl=1*r3b6ks*_gcl_au*MTkzMDY5MjM0Ni4xNzM1NjkyNjU1
(Monarch® DNA Gel Extraction Kit Protocol (NEB #T1020):
https://www.neb.com/en-us/protocols/2015/11/23/monarch-dna-gel-
extraction-kit-protocol-t1020?pdf=true
4. Elute usually 30µl.

DpnI digestion

1. Add the following components to a 1.5ml centrifuge tube.

SN Component Volume
1 PCR product 20µl

2 10X r-Cut Smart 2.33µl

3 DpnI 1µl
2. Water bath the samples at 37˚C for 1 hour.
3. Water bath the samples at 65˚C for 10 minutes to denature the
enzyme.

DNA Clean and Concentrator Kit.

1. Usually use “DNA Clean and Concentrator Kit- 5.” There are 25 ones
too.
2. Follow the protocol: (Zymo Research)
https://files.zymoresearch.com/protocols/_d4003t_d4003_d4004_d4013
_d4014_dna_clean_concentrator_-5.pdf?
_gl=1*1d7og1l*_gcl_au*MTkzMDY5MjM0Ni4xNzM1NjkyNjU1
(Monarch): https://www.neb.com/en-us/protocols/2024/07/16/standard-
cleanup-protocol-using-the-monarch-spin-pcr-and-dna-cleanup-kit-and-
a-vacuum-manifold?pdf=true
3. Elute (usually preferred 30µl) or 13µl.
4. Simple Math that may help (23.3µl * 5 = 116.5 µl )
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Calculate DNA concentration:

1. User Manual: https://www.mt.com/dam/MT-TW/PDFs/%E5%AF

%A6%E9%A9%97%E5%AE%A4%E5%88%86%E6%9E
%90%E5%84%80%E5%99%A8%E6%93%8D%E4%BD%9C
%E6%89%8B%E5%86%8A/%E7%B4%AB
%E5%A4%96%E5%85%89%E8%AD%9C/UV5Nano_en.pdf
2. Clean the Micro Volume Platform thoroughly through pure water.
3. Login to Admin
4. Click dsDNA (By default, it should be OK; if not, reference wavelength:
320nm, Dilution Factor: 1, background correction: 1 point,
Measurement Cell: Micro Volume Platform). Click Start.
5. Add 3µl of water (or Elution Buffer) and click “Measure Blank”
6. Clean the water (or Elution Buffer), and load 3µl sample and click
“Measure Sample.”
7. Record the Sample Concentration in a lab notebook (sample label:
concentration). Also look at the spectrum to verify purity.
8. Clean the Micro Volume Platform. Shutdown.

Gene Block

1. Converter and Reference: https://nebiocalculator.neb.com/#!/od260

2. Centrifuge the Gene Block.

3. Add water (or TE) to make Master Stock (usually 100 µM), and from
there, make Working Stock (usually 10 µM or desired).
4. Record it in Inventory. Store Master Stock at -80˚C and Working Stock
at -20˚C.

Circularization of Gene block

1. For ligation to occur, at least one of the DNA ends (insert or vector)
should contain a 5´ phosphate.
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2. Digestion of DNA with a restriction enzyme will always produce a 5´

phosphate.
3. Follow the protocol, but this is found to be effective for Levenson’s Lab.

S.N. Components Volume

1 Water 6 µl
2 DNA (gene block) 1 µl
3 T4 Buffer of Ligase 1 µl
4 T4 Ligase 2 µl
4. Incubate it at room temperature for 1 hour.
5. Water bath at 65˚C for 10 minutes to denature the enzyme.

RCA (Phi 29XT RCA Kit)

1. Follow the protocol.

S.N. Component Volume
1 Water 10 µl
2 Circularized DNA 2 µl
3 5X Buffer 4 µl
4 10 mM dNTP 2 µl
5 Random Primers 2 µl
6 Mix this (1-5) first and them iso temp (98˚C) for 3 min.
Then, Add DNA polymerase
7 DNA polymerase 2 µl
8 Incubate using Fixed Temperature Cycle in PCR machine
42˚C (2 hrs), Heat Inactivation: 62˚C (10 min), Hold(4˚C)
2. Verify it with 2% agarose gel.

Partial Digestion of Gene Block (SmaI digestion)

1. Set the 2 Water Bath instruments to 37˚C and 65˚C respectively.

2. Add the following components below on ice. The SmaI digestion is a
very fast step. Be quick.

S.N. Component When RCA When RCA When RCA

product is 5 product is product is
µl 7.5 µl 10 µl
1 Water 3 4.5 6
2 RCA product 5 7.5 10
3 10X r-Cut Smart 1 1.5 2
4 SmaI 1 1.5 2
3. Water bath at 37˚C for desired time (usually 30 sec, 1 min, 1:30 sec, 2
min, or 5 min).
4. Denature the enzyme immediately by water bath at 65˚C for 10
minutes.
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Gibson Assembly

1. Note: Usually, in Levenson’s Lab, we do not add water and scale the
reaction down to a total of 10µl (5 µl 2X HiFi DNA assembly Master Mix
and 5 µl of Vector and Insert).
2. Protocol: https://www.neb.com/en-us/protocols/2014/11/26/nebuilder-
hifi-dna-assembly-reaction-protocol
3. NEB recommends a total of 0.03–0.2 pmols of DNA fragments when 1
or 2 fragments are being assembled into a vector, and 0.2–0.5 pmols
of DNA fragments when 4–6 fragments are being assembled.
4. Record the concentration and basepair length of vector and insert
5. Converting (ng/µl= µg/ml) to pmols/µl
(weight ∈ng)∗1000
pmols=
(base pairs length of fragment )∗(650 daltons)

for reference :50 ng of 5000 bp is about 0.015 pmols

6. Add 5 µl of 2X HiFi DNA assembly Master Mix.

7. Add a total of 5µl of vector and insert in a 1:2 ratio, i.e., vector: insert
= 1: 2 of pmols.
8. Incubate (water bath) the sample at 50˚C for 1 hour.
9. Cool the sample before Bacterial Transformation or store it at -20˚C for
future use.

Agar Plate

Luria agar contains agar, a solidifying agent, which allows it to solidify into a
solid medium on a petri dish, while Luria broth remains liquid and is used in
tubes for bacterial culture.

Making LB agar:
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1. Dissolve completely 40-gram Luria agar in 1 L water by keeping it on a

sitting plate. (Usually, use 32 g for 760 ml water to distribute 200 ml to
four 250 ml bottles.)
2. Loosen the cap of the bottles and autoclave the bottles in the liquid
cycle.
3. Close the cap airtight and let it cool down to Room temperature or
50˚C (if making agar plates). It will solidify at room temperature.

Making Agar Plates:

1. Let LB agar cool down to 50˚C, or microwave the LB agar if solidified

LB agar is present. Microwave until there is no solidifying chunk left.
Remember to loosen the cap. Then cool down to 50˚C.
2. Add antibiotics and mix by swirling. (for reference, if 1000X kanamycin
stock is present, add 200µl of Kanamycin for 200 ml of LB agar).
3. Pour it into the plate and let it solidify at Room Temperature.
4. Store it at 4˚C.

LB media

1. Dissolve completely 25-gram Luria broth in 1 L water by keeping it on a

sitting plate. (Usually, use 12.5 g for 500 ml water to distribute equally
into three 250 ml bottles)
2. Loosen the cap of the bottles and autoclave the bottles in the liquid
cycle.
3. Close the cap airtight and let it cool down to Room temperature.

Bacterial Transformation

For minipreps, use NEB 5α competent cells; for protein expression, use
BL21 cells.
1. Grab Ice
2. Thaw the cells on ice. Take the agar plate out of the fridge.
3. Add plasmid to the thawed cells and flick it 4-5 times; do not vortex.
Usually, a maximum of 10% to the cell volume (for, e.g., 2µl plasmid in
20 µl cells) or (cells: plasmid ratio = 9:1 (by volume)).
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at exactly 42°C for exactly 30 seconds (NEB 5 α) or 10
seconds (BL21).
6. Place on ice for 2 to 5 minutes.
7. Add 700 µl SOC media to the cell tubes and incubate it at 37˚C in a
shaking incubator for 45 minutes.
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8. Centrifuge the tubes at 250 rpm ~ 1000* g for 5 min (yes, thousand g
only).
9. Remove 600µl of media.
10. Resuspend the cells in the remaining media and transfer them
into agar plates.
11. Shake the agar plates with glass beads. Incubate it at the desired
temperature (usually 37˚C).
12. Make sure to autoclave glass tubes; you will need them in the
next step.

Can I do overnights without incubating bacteria in an agar plate?: Yes, but

do not do it. You risk the chance of contamination.

Overnight

1. Add 5 ml of LB broth to glass tubes.

2. Add 5 µl of Antibiotics (1000X) to glass tubes.
3. Pick the colony by pipet and drop it in the glass tube.
4. Incubate overnight (12-16 hrs) in a shaking incubator at 37˚C.

Making Autoinduction Media

Making autoinduction Media

1. Add the following components to the desired water volume.

S.N Component 0.25L 0.5L 1L 1.5L

.
1 Phosphate Buffer pH 1.5 g KH2PO4 3g + 6g 6g + 9g + 18g
(7.2) + 3g Na2HPO4 12g
2 NaCl 2.5 g 5g 10 g 15g
3 Yeast Extract 2.5 g 5g 10 g 15g
4 Tryptone 10g 20 g 40g 60g

Overnight in Autoinduction Media

1. Remember to do general overnight before Autoinduction media.

2. Add the following components and incubate overnight (12-16 hrs) in a
shaking incubator at 37˚C

Items 5ml 500ml 1L

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Autoinduction 5 ml 500 ml 1L
media
Glycerol (60% v/v) 50µl 5ml 10ml
Glucose (10% w/v) 25µl 2.5 ml 5ml
Lactose (8% w/v) 125µl 12.5ml 25ml
Antibiotic (1000X) 5µl 500µl 1ml
Overnight 25µl 1ml 1ml

Miniprep

1. Transfer 1.5 ml overnight to a centrifuge tube. Centrifuge it at

16000*g. Discard the supernatant. For more DNA, add 1.5 ml again
and centrifuge again.
2. Follow the protocol, Usually Elution of 30µl : https://www.neb.com/en-
us/protocols/2024/04/16/plasmid-miniprep-protocol-using-
centrifugation-t1110
3. Calculate the concentration of DNA.

Sanger Sequencing

1. Add 5 µl Forward Primer (T7) and 5 µl DNA to the sequencing tube.

2. Record the Code of the Sequencing tube and corresponding DNA in
your lab notebook; you will have to figure out which one is which
yourself by referencing the code of the tube.
3. Give Prof Levenson the Sticker barcode and corresponding sample
identity and he will order it.
4. Place samples into Eurofins sequencing baggie and label bag “Sanger
Sequencing”.
Email: rlevenson@soka.edu
Phone No: 949-269-5510

Whole Plasmid Sequencing

1. Add 10μl of DNA (usually greater than 30μg/mL) in a 1.5 centrifuge

tube. You do not need to add Primers for Whole Plasmid Sequencing.
2. Place the Eurofins sticker barcode on the side of the tube. Any label on
top of tube is just for your/our reference and not read by Eurofins.
3. Give Prof Levenson the Sticker barcode and corresponding sample
identity and he will order it.
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4. Place samples into Eurofins sequencing baggie and label bag “Whole
Plasmid Sequencing”.

Cell Lysis

1. Transfer 1 ml bacterial cells from the autoinduction media to the

centrifuge tube and centrifuge 5000*g for 10 minutes.
2. Aspirate the supernatant.
3. Resuspend the pallet in 200µl B-PER reagent and incubate for 15
minutes at room temperature.
Meanwhile, make SDS-PAGE Master Mix. Make two volumes of Master
Mix per cell construct.

S.N Component Volume (Total: 64

. µl)
1 SDS-PAGE loading 40µl
dye
2 5mM DTT 16µl
3 Water 8µl

4. Centrifuge it at 16,000* g for 5 minutes.

5. Transfer the supernatant (soluble fraction) to the new centrifuge tube.
Label it as “Sol _____”
6. For pallet (insoluble fraction), resuspend the pallet in 100µl water.
Label it as “Insol _____”
7. Take a new test tube and load 16µl of Sol or Insol fraction with 64µl
SDS-PAGE Master Mix.
8. Heat the tubes with (sample and Master Mix) at 90˚C for 10 min;
remember to vent the tubes. Allow it to cool, then store it in -20˚C or
run SDS-PAGE gel.
9. Right before loading the gel, centrifuge each sample to be loaded at
15,000 × g for 3 min. Load 8 μL of the “Load” samples into each well.
When loading “Insol” fractions, take your sample from the top of the
tube, as there may be a viscous precipitate at the bottom. Load 3 μL of
the ladder (NEB Color Prestained Protein Standard, Broad Range).

Buffers for SDS-PAGE

Tris Buffer (1M , 1L, pH: 8.8)

Cb
pH= pKa+log ( )
Ca
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Cb
¿ , 8.8=8.07+ log ( )
Ca

Cb
¿ , 5.370=log ( )… … … . eqn (i)
Ca

Now, We are making 1M solution in 1L. So,

1 L∗1 M =1 mole, so

C a +Cb =1 … … … . eqn(ii)

From eqn(i) and (ii)

Ca = 0.156 moles

Cb = 0.837 moles.

So, we should add 0.156 moles of acid and 0.837 moles of base in 1 L
of water. (Tris.HCl+ Tris.Base)

0.156 moles of Tris.HCl = 24.59 g

0.837 moles of Tris.base = 101.4 g

Adjust the pH by adding NaOH or HCl.

Tris Buffer (1M, 1L, pH: 6.8)

0.949 moles of Tris.HCl = 149.56g

0.0509 moles of Tris.base = 6.174 g

3.5x Bis-Tris Buffer (1.25M, pH: 6.5)

Make 1.25 M in 50ml buffer and adjust the pH with HCl.

So Molecular weight = 209.24g/mol

We know,

Mass(g) = Molarity (M) * Volume(L) * Molecular Weight (g/mol)

= 1.25* 0.05* 209.24

=13.0075g

1 M Stock solution of Sodium Bisulfite (Mild Reducing Condition)

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MW: 104.061 g/mol

We will make 10 ml of 1M stock solution.

We know,

Mass(g) = Molarity (M) * Volume(L) * Molecular Weight (g/mol)

= 1 M * 0.01L* 104.061g/mol

= 1.04061g

Therefore, we will add 1.04061 g of Sodium Bisulfite in 10ml water to make

1M solution.

Now, we should make final conc of 5mM in a running buffer. So,

M 1∗V 1=M 2∗V 2

1 M∗X =0.005 M∗700 ml (imagining the running buffer is 700ml)

X =3.5 ml

Therefore add 3.5 ml of the Sodium Bisulfite to the running buffer. Always
add fresh.

SDS- PAGE GEL

Making/ Casting SDS-PAGE Gel

Be Careful: TEMED is a neurotoxin.

1. Mix the component of Resolving Gel first in a 15 ml conical tube and
pour it between the Glasses. Add 1 ml of (water or preferred:
isopropanol) to create an even surface and let the gel polymerize.
Then, Mix the components of Staking Gel in a 15 ml conical tube and
pour it between the glasses on top of the Resolving Gel. Add a comb
and let it polymerize.
2. It can be stored at 4˚C for a few days. Cover the whole gel with the
comb wrapped, with wet water paper and store it in the fridge.
a. Bis-Tris Gel (

Use MES when you want to separate low molecular weight

proteins (≤50kDa).

Use MOPs when you want to separate high molecular weight

proteins (≥50kDa)).

10% Bis_Tris 4% Bis_Tris Buffe

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Resolving Stacking rs
1 Gel 2 Gels 1 Gel 2 Gel MES
835 MOP
30% Acrylamide/Bis (*2) 835 (*4) 230 460 S
710
3.5x Bis-Tris buffer (*2) 710 (*4) 500 1000
955
H2O (*2) 955 (*4) 510 (*2) 510 (*4)
10% APS 25 50 20 40
TEMED 7 14 10 20

b. Tris-Glycine Gels
12% 4% Tris_Glycine Buffe
Tris_Glycline Stacking rs
Resolving
1 Gel 2 Gels 1 Gel 2 Gel
30% Acrylamide/Bis 1000 1000 260 520
(*2) (*4)
1 M Tris pH pH pH pH
(8.8) (8.8) (6.8) (6.8)
925 (*2) 925 250 500
(*4)
10% SDS 50 100 20 40
H2O 525 (*2) 525 725 725
(*4) (*2) (*4)
10% APS 50 100 20 40
TEMED 5 10 2 4
Running an SDS-PAGE Gel

1. Fill the inner chamber and outer chamber with the buffer
(MES/MOPS/Tris-Glycine Buffer). Remember you will have pairs of gels
or need a buffer dams.
2. Load the ladder and the sample in the wells.
3. Run the gel at 200 V until the blue reaches nearly the bottom of the gel
(typically, from 34 min to an hour).
4. Gently (it tears very easily) remove the gel and keep it in a gel box (for
Staining ) or directly to the sandwich (Western Blot).
5. For staining, add about ~20 ml of PAGE BLUE to the gel and keep in the
rocker for 1 hour or (overnight). To destain, discard the Page Blue in a
disposable container (not in the sink because it stains everything.) Add
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water and keep in the rocker for 5 min; change the water again and do
so until necessary. Then, image the gel.

Western Blot

Day 1

1. Keep the black-facing part in the tray and follow the order from step 1
to step 6.
2. Soak Sponges (black and white with) transfer buffer and place it on
black-facing part.
3. Put SDS-PAGE gel and then put membrane above SDS-PAGE gel.
4. Activate the membrane with Methanol. Reuse methanol. Remember:
Methanol cannot go in a sink.
5. Put filter paper, then sponge and assemble the cascade and keep in
blot frame.
6. Add transfer buffer to the blot chamber tank and remember to put ice
bag.
7. Run it at 60 V for 1 hour. Find out the necessary dilution of primary
antibody and secondary antibody. Do not dilute them in water.

8. Dissolve 5% milk in TBST. (for reference: 2 g milk in 40 ml TBST).

9. Keep the membrane in a tray and block the membrane by pouring
about 25~30ml (5% milk/TBST) and keep in rocker for 30 minutes.
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Discard the milk. Meanwhile, Make primary antibody dilutions in

5%milk/TBST solution.
10. Add the primary antibody solution to the tray containing
membrane (close the lid) or in a membrane enclosed plastic (seal it)
and leave it overnight at 4˚C.

Day 2

1. Wash with 1X TBST thrice for 5 min each. Meanwhile make secondary
antibody dilution in 5%milk/TBST solution (only TBST is fine too).
2. Place the secondary antibody in membrane and keep in rocker for 30
min. Discard the solution after that.
3. Wash once with TBST for 5 min then once with TBS for 5 min.
4. Prepare 1ml ELC by 1:1 ratio of subunits).
5. Place the ELC in the imager tray (Blot/UV/Stain-Free Sample Tray).
Place Membrane and then flip the membrane both side to ensure the
ELC has spread all over, Place the protein facing part upside and image
the membrane.