MBIO 3600 - Protocols

Things to keep in mind during dilutions

1.) After each transfer, mix the tube by vertexing

2.) When transferring 100 μl use P200, but when transferring 900 μl, use P1000. 

Things to keep in mind when plating dilutions

1.) Aseptically transfer 100 μl onto plate

2.) Use a disposable spreader for each dilution 

Plating factor

Reciprocal of volume plated

Dilution factor

Reciprocal of dilution for significant counts 

Significant plate counts

Sum of the plate counts at significant dilutions / number of significant plates

Accurate counts

Number of non-overlapping colonies

Significant colony counts

The typical accepted range is 30 to 300, BUT this range should be based on knowledge and experience with the particular microbe 

More than 300 colonies (When are they significant?)

They are significant as long as the colonies are non-overlapping and they are accurately counted

More than 300 colonies (Why are they not always counted?)

Competition for nutrients between closely spaced colonies causes viable cells that CAN grow to not form a colony, due to limited amounts of food.

Less than 30 colonies (When are they significant?)

(1) If it is the highest counts possible
(2) If certain studies, such as inhibition studies generally result in a low colony count, simply due to the nature of the experiment.

Titre calculation 

"
THEN AVERAGE AT THE END
"

What are outliers in plate counts?

Any significant plate count that does not follow the expected pattern, usually when there is a 10-fold difference.

Gram stain steps

1.) Prepare a smear of bacteria and heat fix onto slide

2.) Stain with crystal violet for 1 minute and then rinse with water

3.) Add iodine solution and let sit for 1 minute

4.) Wash iodine off with water and then add acetone until free of colour

5.) Wash slide with water and blot dry

6.) Stain slide with safranin and then wash 

Purple cells in gram stain = 

Gram positive

Pink cells in gram stain

Gram negative

Use of hemocytometer (STEPS)

1.) Remove and wipe hemocytometer from sealed container, using only one of the inlets on either side

2.) Transfer 10 μl to the V-shaped grove on the hemocytometer

3.) Place on microscope and then count the regularly-shaped microorganisms using the 40x objective

4.) Count the number of organisms in rows 1, 2, 4, and 5 (if they overlap rows, only count them ONCE)

5.) Hemocytometer Titer Calculation

Hemocytometer Titer Calculation

"
Ex.) 
"

Setting up agarose gel 

(1) Ensure gel tray is level and that upper notches are close to the negative electrode

(2) Place the black gates securely into the slots

(3) Place comb into upper notches

(4) Add 30 ml of agarose 

When would you want to tape the wells?

It’s usually when you’re eventually doing a gel isolation and want ALL of the DNA to be in the gel.

Choosing the percentage of agarose

Higher percentages are usually used to separate smaller fragments 

When putting in the agarose, what is something you want to make sure to prevent?

Avoid bubble formation.

What do you if bubbles form

Remove them with a pipetman or move them to edge of the tray

Loading + running gel steps

(1) After gel has solidified, remove comb and gates

(2) Pour 1x TAE buffer just below the fill line, making sure some goes into the well

(3) Add appropriate amount of dye to DNA sample 

(4) Load the first well with the 1-10μl of the ladder and then load the sample into the other wells

(5) Run the gel at 100 volts for about 25 minutes 

What direction does the DNA travel in a gel electrophoresis

From the negative to the positive (since the DNA is negatively charged)

Three main ways of visualizing DNA on gel

1.) The DNA staining dye is present in the loading buffer

2.) The staining dye is present in the gel itself

3.) The DNA is soaked in buffer containing a DNA staining dye, AFTER the gel has run

DNA staining dyes

Makes the DNA fluoresce under UV light, so we can visualize the DNA, but they are not visible with the naked eye

Tracking dyes 

1.) Can be seen with the naked eye

2.) Migrates along with the electric current, allowing us to determine when the gel is done running

3.) Usually want it to be as close to the bottom as possible, especially if the we’re trying to visualize very small DNA fragments 

Loading buffers

They contain a high density agent that allows the sample to sink into the well, as well as a tracking dye (the DNA staining dye may or may not be included). 

Do loading buffers always have both a staining dye and tracking dye

No, but they always have a tracking dye

EZ-vision

1.) 6x loading buffer with a fluorescent DNA staining dye

2.) Non-mutagenic and non-toxic

3.) Stop the run when the tracking dye is NEAR the bottom (not necessarily at the bottom)

FastDigest green buffer (for gel) 

1.) It contains a density reagent that causes sample to sink to the bottom of the well

2.) Has two tracking dyes: blye one that migrates at 3kb and a yellow one that migrates at 10bp

3.) Stop the run when the yellow tracking dye is AT the bottom

4.) It has NO staining dye, instead this is usually added to the gel itself (such as GelRed)

GelRed

A nucleic acid gel strain present IN THE AGAROSE GEL that visualizes the DNA, such that it is NOT a tracking dye but there must still be a tracking dye in the loading buffer.  

Gel extraction + purification steps

(1) Cut out band from gel and put into microcentrifuge

(2) Mash up with a wooden stick and estimate the gel volume

(3) Add solubilization buffer 

(4) Let dissolve in 50ºC water bath, mixing every couple minutes

(5) Add isopropanol

(6) Add into spin column and centrifuge

(7) Add in Wash buffer + centrifuge

(8) Add in ethanol + centrifuge

(9) Add in elution buffer (water) --> KEEP ELUTION 

Addition order of reagents in gel extraction + purification 

1.) Solubilization buffer

2.) Isopropanol

3.) Wash buffer

4.) Ethanol

5.) Elution buffer

What is the solubilization buffer for (gel extraction)

1.) Has a pH indicator that ensures the pH is below 7.5, as binding to the silica is pH dependent

2.) Has a chaotropic agent that solubilizes the agarose when heated

3.) Contains high salts which helps the DNA bind to the silica, as it neutralizes the repulsion of the phosphate groups of DNA 

What is the isopropanol for? (gel extraction)

It solubilizes the dNTPs, which helps exposes the phosphate groups and therefore, helping with the binding to the silica

What is the wash buffer + Ethanol for (gel extraction)

1.) They help wash away the unwanted products (i.e. primers, enzymes, salts), while the desired nucleic acid remains bound to the silica.
2.) The ethanol also helps elute any residual water from the membrane.

What is the elution buffer for? (gel extraction)

It is to elute the DNA from the column

Genomic DNA isolation (STEPS)

1.) Prep lysate

2.) Binding step 

3.) Wash DNA

4.) Elute DNA 

Prep lysate steps (gDNA isolation)

(1) Pellet, then resuspend the cells in PBS

(2) Add proteinase K and RNaseA

(3) After incubation, add lysis/binding buffer

(4) Incubate and then add 100% ethanol to lysate

Binding step (gDNA isolation)

Transfer to spin column

Wash DNA step (gDNA isolation)

(1) Add wash buffer 1 + centrifuge

(2) Add wash buffer 2 + centrifuge 

Elute DNA step (gDNA isolation)

(1) Place into collection tube

(2) Add elution buffer 

What is PBS for?

A buffer that resuspends pellets cells, optimized specifically for tissue lysis in the presence of proteinase K

What is proteinase K for?

It digests proteins, releasing genomic DNA from proteins and cellular components in the process

What is the RNAseA for?

It degrades any RNA, ensuring that the only thing left intact is the DNA

What is the ethanol for? (gDNA isolation)

It provides the optimum conditions for the DNA to bind to the silica solid phase

What is the wash buffer 1 and 2 for? (gDNA isolation)

It contains ethanol and salts, which helps remove enzymes and proteins, as well as helps the DNA better bind to the silica (specifically the ethanol) 

Why have two different wash buffers? (gDNA isolation)

The first one helps remove the main contaminants, such as proteins, while the second one helps remove any residual contaminants, such as salts.

What is the elution buffer for? (gDNA isolation)

To elute the DNA from the column

X-gal detection system (blue vs. white colonies)

1.) Blue colonies = Functional LacZ enzyme, meaning there is NO insert in the MCS

2.) White colonies = Non-functional LacZ enzyme, because there IS an insert in the MCS

Plasmid DNA isolation begins with…

Growing the bacteria that holds the plasmid to create multiple copies and then selecting for only those that hold the plasmid by plating them onto the antibiotic the plasmid is resistant to

Plasmid DNA isolation principle

It is based on the fact that alkaline conditions will lead to the degradation of chromosomal DNA, but it closed circular plasmids are able to return to their normal configuration when the pH is brought back to normal.

Two main steps of plasmid DNA isolation

1.) Extraction of plasmid DNA
2.) Purification of plasmid DNA

Addition of reagents (gDNA isolation) 

1.) PBS
2.) Proteinase K + RNase A
3.) Lysis/binding buffer
4.) 100% ethanol
5.) Wash buffer 1
6.) Wash buffer 2
7.) Elution buffer

Extraction of plasmid DNA (steps)

(1) Pellet cells

(2) Resuspend cells in resuspension solution, containing RNaseA

(3) Add lysis solution

(4) Add neutralization solution

(5) Centrifuge --> To pellet the cell debris and chromosomal DNA

(6) KEEP the supernatant, as it contains the plasmid, and transfer to spin column 

Purification of plasmid DNA (steps)

(7) Centrifuge

(8) Add wash solution + centrifuge (3x)

(9) Add elution buffer into collection tube 

Addition of reagents (Plasmid DNA isolation)

1.) Resuspension solution (w/ RNase A)
2.) Lysis solution
3.) Neutralization solution
4.) Wash solution
5.) Elution buffer

What is the resuspension solution for? (plasmid DNA isolation)

Resuspends cells homogenously, in a way that it is appropriate for cell lysis

Components of resuspension solution (plasmid DNA isolation)

1.) Tris-HCl

2.) pH 8.0

3.) EDTA

4.) RNase 

Tris-HCl, as well as pH 8.0 (resuspension solution in plasmid DNA isolation) 

Ensures optimum ionic and pH stability of cells 

EDTA (resuspension solution in plasmid DNA isolation) 

It chelates divalent cations that may be involved in the initial destabilization of cell walls, but it DOES NOT lyse the cell AND it inactivates DNases. 

RNase (resuspension solution in plasmid DNA isolation) 

Degrades RNA, since we only want plasmid DNA

What is the lysis solution for (plasmid DNA isolation)

It provides alkaline conditions that denatures the DNA, but it lyses the cells in a way that the cell membrane remains attached to the genomic DNA, so that when conditions are neutralized, the cell debris and gDNA are precipitated out together

Components of lysis solution (plasmid DNA isolation) 

1.) NaOH
2.) SDS

NaOH in lysis solution of plasmid DNA isolation

It provides alkaline conditions, resulting in the denaturation of both genomic and plasmid DNA into single strands

SDS in lysis solution of plasmid DNA isolation

Lyses the cells by dissolving the cell membrane and solubilizing the phospholipids

What is the neutralization buffer for (plasmid DNA isolation)

It neutralizes the alkaline conditions so that cell debris + genomic DNA precipitates, while plasmid DNA remains in solution

Components of neutralization buffer (plasmid DNA isolation)  

1.) High concentration of potassium acetate + pH 4.8

2.) Guanidine hydrochloride 

High concentration of potassium acetate + pH 4.8 (neutralization buffer in plasmid DNA isolation)  

It precipitates out proteins and other cell debris, including gDNA, such that they precipitate out together. 

What is the high salt concentration for? (neutralization buffer in plasmid DNA isolation)  

The high salt concentration allows for the binding of plasmid DNA to the silica membrane

Guanidine hydrochloride (neutralization buffer in plasmid DNA isolation)  

It denatures proteins and inhibits DNase activity, as well as enhance DNA binding to silica membrane

What is the wash buffer for? (plasmid DNA isolation) 

1.) It helps remove any remaining nuclease activity and other impurities, while keeping the plasmid DNA attached to the silica membrane.
2.) It also contains EDTA that helps prevent DNA degradation

After adding the lysis solution, what do you want to avoid and why (plasmid DNA isolation)

Avoid vortexing, because it might shear the chromosomal DNA

After adding the neutralization solution, what do you want to do and why (plasmid DNA isolation)

Mix gently to avoid localized precipitation of bacterial cell debris. 

PCR purification steps 

(1) Add binding buffer (with isopropanol) to PCR product

(2) Mix thoroughly, then transfer to spin column + centrifuge

(3) Add wash buffer + centrifuge

(4) Centrifuge again

(5) Add elution buffer into collection tube  

Addition of reagents during PCR purification

1.) Binding buffer
2.) Wash buffer
3.) Elution buffer

What is the binding buffer for? (PCR purification) 

It contains high salt concentration + appropriate pH + isopropanol that allows the PCR product to bind to the silica gel membrane 

What is the wash buffer for? (PCR purification)

It contains ethanol that helps remove contaminants, such as primers, nucleotides, enzymes, etc. 

Competent cells require treatment with…

1.) Calcium

2.) Magnesium

3.) Temperature near 0ºC

Treatment of cells with calcium, magnesium and a temperature of 0ºC promotes what?

It creates competent cells that promotes the uptake of DNA during transformation

What is heat shocking for during transformation

It facilitates complete DNA molecule uptake by the cells

Why must the cells be incubated in media before they are plated with the antibiotics?

1.) It is so the plasmid can establish themselves in the cell.

2.) It allows plasmid antibiotic resistant genes to be expressed prior to exposure to antibiotics, as it increases the survival of the bacteria on the antibiotic containing plate 

Two steps of making and transforming compoetent cells

1.) Preparation of competent cells
2.) Transformation of competent cells

Preparation of competent cells steps

(1) Inoculate broth with overnight E. coli culture and then rotate for 3 and a half hours

(2) Centrifuge cells and then resuspend with sterile MgCl2

(3) Centrifuge and then resuspend in sterile CaCl2

(4) Incubate on ice for 30 minutes and then centrifuge

(5) Resuspend cells in sterile CaCl2 + glycerol 

Addition of reagents in the preparation of competent cells 

1.) MgCl2
2.) CaCl2
3.) CaCl2 + glycerol

Stransformation of competent cells steps

(1) Remove a portion of competent cells for the negative control

(2) Heat shock at 43ºC for one minute

(3) Add nutrient rich broth and then incubate for an hour

(4) FINALLY, spread plate transformation mixture onto plate with appropriate antibiotic 

During the preparation of competent E. coli cells, why do we have to gentle when mixing + centrifuging at slow speed

To prevent accidentally killing any cells 

Primer PAIR design requirements"

1.) Sizes should be 16 to 22 bp, but pair does not differ by more than 3 bp

2.) Tm should be 40 to 60ºC, but pair does not differ by more than 5ºC

3.) GC content should be 35 to 60%

4.) 3’ end should be either G or C

5.) Should have NO inverted repeats (unless the Tm is low, then it’s okay because then hairpins will not form)

Tm calculation if more than 14 bp

"

Tm = 64.9ºC + 41ºC x ((# G + C’s in primer – 16.4)/N) --> N = length of primer

"

Tm calculation if less than 14 bp

Tm = 4ºC x (G+C) + 2ºC x (A+T).

The template DNA in a PCR should be suspended in…

Low EDTA

Phusion Flash II DNA pol

DNA pol that have DNA pol activity, but also 3’ to 5’ exonuclease activity, and it allows for rapid amplification of DNA 

Special abilities of Phusion Flash II DNA pol 

It inhibits DNA pol activity at room temperature AND blocks its 3’ to 5’ exonuclease activity during the reaction to prevent degradation of primers and template DNA. 

Reaction components in a PCR

1.) dNTPs in equal molar amounts

2.) 1.5 to 2.5 M MgCl2

3.) Reaction buffer, usually with Tris-HCl

Mutant primer requirements 

1.) 16 to 25 bp

2.) Low melting temperatures (42 to 48ºC)

3.) Mutation occurs in the middle, with 8 to 10 bp on either side

Exterior primer requirements (Oligonucleotide-directed Mutagenesis Primer Design) 

1.) Reverse primer in PCR 1 should have a low Tm + LOW annealing temp + SHORT

2.) Forward primer in PCR 2 should have a high Tm + HIGH annealing temp + LONG

Why is the annealing temperature of the forward primer higher than the reverse?

To prevent annealing of any residual reverse primers left over from the first reaction.

SDS-PAGE steps

(1) Load 40 μl of sample + 10 μl of loading buffer --> Heat at 95ºC --> let cool before loading

(2) Load 10 μl of the sample + buffer

(3) Load 20 μl of the protein standard

(4) Attach to machine and run at 200 volts for 35 minutes

(5) ENSURE THAT THE TRACKING DYE DOES NOT RUN OFF THE GEL

(6) Remove glass plates and float gel off in the buffer

(7) STAIN if not doing western blot

Electrophoretic Transfer to Nitrocellulose Blot steps

(1) Cut membrane to be the same size as the gel and is wet with transfer buffer

(2) Also saturate the filter papers and fibre pads

(3) Assemble cassette in order, starting from the black side panel (negative side)!!!

(4) Place Ice pad next to negative cathode

(5) Run at 75 volts for 20 min