All experiments

Experiment 1

Introduction:

In this experiment, mutagenic primers can be used to introduce mutations into plasmids using PCR. This experiment aims to introduce a specific mutation in the nucleotide sequence of the phoA gene contained in the plasmid pJR-1U using primers known as site-directed mutagenesis (SDM). The next goal of this experiment is also to transform these mutated plasmids into the JW0374 strain of E. coli. JW0374 cells.

Mutagenesis and DPNL2 Digest

Mutations: K328A and D101N.

• Sufficient mutagenesis master mix for 2.5 reactions. Every 47 µL of the master mix will contain:
• 50 ng DNA template
• 1 U KOD Hot Start polymerase
• 1X KOD Hot Start buffer
• 1.5 mM MgSO₄
• 0.2 mM dNTPs
• Enough water to bring the total volume to 47 µL
• Mutagenic oligonucleotide primers (15 pmol of each primer in 1.5 µL)
• 2 microcentrifuge tubes (0.2 ml)

mix thawed solutions thoroughly

K328A

D101N

You will carry out two thermocycler reactions: one reaction containing the mutagenic primers and pJR-1U, as well as a one containing pJR-1U, but no primers.

Set up two microfuge tubes in the ice bucket. Label one tube with the name of your mutant and one tube “control,” In these tubes, prepare reactions according to the following recipe indicated below.

Gently mix each reaction by pipetting the solution up and down several times.

Place your samples in the thermal cycler.

Upon completion of temperature cycling, place your microfuge tubes on ice for 2 minutes to cool the reaction below 37 °C

Reserve 5 µL of each reaction in an appropriately labeled tube for electrophoresis.

Add 1 µL of DpnI endonuclease to each tube (using the supplied 2 µL pipetman) and gently mix the reaction by pipetting up and down several times.

Incubate your reactions at 37 °C for 25-30 minutes.

Prepare for the transformation while the DpnI digest is running. Following the digest, reserve 5 µL of each reaction in an appropriately labeled tube for electrophoresis.

Store all the samples that you’ve made thus far in your box in the freezer.

Transformation

The mutated pJR-1U plasmid will be transformed into the JW0374 strain of E. coli. Therefore, treat these cells gently and to keep them chilled at all times.

Obtain the following:

30 µL of chemically competent cells

1 ml SOC media

1 sterile, disposable culture tube

1 LB/Amp plate

Get the LB/amp plate (no cells in it) and let it warm to room temperature

label it with our section number, the date, initaials, and intended mutation or control conditions

Thaw the competent cells on ice.

Add 30 µL of competent cells to 3 µL of your DpnI-digested mutagenesis reaction (not the control reaction).

The TA will do the same thing with one group’s control reaction, as well as with 1 µL of diluted pJR-1U stock.

Incubate the cells and DNA on ice for 30 minutes.

Move the cells to the heat block set to 42 °C for 45 seconds.

Place your cells and DNA back on ice for 2 more minutes.

Use a P-200 pipetman to transfer the transformed cells and media to a sterile culture tube.

Add 1 mL SOC to the tube.

Incubate the cells in the shaking incubator at 37 °C for 1 hour.

Transfer your culture to a 1.5 mL Eppendorf tube and spin down in the microfuge. 5 minutes 15k rpm.

Remove 800uL of the SOC from the tube (leaving the pellet intact) and resuspend the pellet in remaining culture (in the remaining 200uL of SOC after removing 800uL)

Spread 200 µL of the transformed, rescued cells on an LB/AMP plate (100 µg/ml ampicillin) until the SOC has been absorbed into the agar.

Place the plate in the incubator with the agar side up, and grow the cells overnight at 37 °C.

Waste:

1) Excess cells should be placed in the “cell waste” in the waste disposal hood. This disposal container is for liquid cell waste only.

2) Non-glass items contaminated with cells go in the orange biohazard waste bag. This is not the same as “anything you used during this lab.”

3) Glass waste is always disposed of in the glass waste disposal box. Only glass waste goes in the glass waste disposal box. Clear plastic is not glass.

Conclusion:

3 control plates

1 plate with only JW0374 Cells no plasmid

Experiment 2

Introduction

29275352.pdf

This experiment aims to isolate the plasmid DNA from the previously transformed bacteria of the mutagenesis reaction. The second goal of this experiment is to determine whether any of the clones include the expected mutation by screening for a change in the digestion pattern of a DNA restriction endonuclease.

Procedure

Plasmid Mini Prep

PDF Attachment is full protocol

Transfer 1.5 mL from a fresh overnight culture to a microcentrifuge tube. To pellet bacteria, centrifuge at full speed (16 000 × g) in a microcentrifuge for 30 seconds. Discard supernatant and re-centrifuge. Remove any residual supernatant using a pipette

Add 175 μL Lysis buffer type 7 to the bacterial pellet and thoroughly re-suspend the pellet.

Add 175 μL Lysis buffer type 8 and mix immediately by gentle inversion (approximately 5 times) until solution becomes clear and viscous.

Add 350 μL Lysis buffer type 9 and mix immediately by gentle inversion until the precipitate is evenly dispersed.

Centrifuge at full speed (approximately 16000 × g) for 4 minutes.

During centrifugation, for each purification that is to be performed, place one illustra plasmid mini column in one Collection tube.

Carefully transfer the cleared supernatant to the mini column (approximately 700 μL). Close the lid of the column gently.

Centrifuge at full speed (approximately 16000 × g) for 30 seconds. Discard the flowthrough by emptying the Collection tube.

Wash the column with 400 μL Lysis buffer type 9 and centrifuge at full speed (approximately 16000 × g) for 30 seconds. Discard the flowthrough.

Add 400 μL Wash buffer type 1 to the column and centrifuge at full speed (approximately 16000 × g) for 1 minute. Carefully discard flowthrough and the Collection tube.

Transfer the illustra plasmid mini column into a fresh microcentrifuge tube and add 100 μL Dh2o type 4 directly onto the center of the column.

Incubate the column for 30 seconds at room temperature.

Microcentrifuge at full speed (approximately 16000 × g) for 30 seconds to recover the plasmid DNA as flowthrough in the microcentrifuge tube.

Store the purified plasmid DNA at -20°C.

Protein Quantification

• Determine concentration and purity of DNA at 260 nm and 280 nm with background correction at 320 nm.
• Use cuvettes with broader wavelength range
• Dilute 5 uL of DNA to 100 uL final volume with water +700uL water

	WT	M
260	0.313	0.319
280	0.188	0.189
320	0.126	0.126

Restriction Digest

Endonuclease used: FSP I

FSP I has 10U per uL

~1

You will set up restriction endonuclease reactions by first preparing a cocktail of buffer and enzyme for multiple reactions (a master mix). For purposes of consistency, make more master mix than you need – but only 1 or 0.5 reactions too much. Then add an aliquot of this mixture to an aliquot of your DNA.

For many enzymes, 1-3 U are required to completely digest 1 µg of DNA in 1 hr. You will digest your samples (your mutated DNA and 1 wild type control), each containing approximately 1 µg of plasmid DNA. The instructions below have you make enough Master Mix for 3 samples (just in case), meaning you should use between 3 and 9 U total.

Obtain the appropriate 10X reaction buffer (and BSA, if required). Set up microfuge tubes and label them with the name of each sample (D153N-1, etc for mutants, (+) for positive control—digested WT, and (−) for negative control—undigested WT). Reserve a tube for mixing your master mix (keep the master mix on ice until you add it to your digest reactions).

Add the enzyme to the Master Mix last. Gently mix by pipetting slowly up and down or by flicking the bottom of the tube with your finger. Do not vortex! Centrifuge the tube briefly if solution splashes up on the side of the tube. Keep this on ice until you’re ready to use it.

Add Master Mix to each mutant DNA sample and the WT positive control. Do NOT add Master Mix to your negative controls! Instead, add water to bring the volume of your negative controls up to 10 µL. Place tubes in the heat block at the optimal temperature for your enzyme, and incubate them for 1 hr. While waiting, prepare an agarose gel.

Remove tubes from the heat block and spin briefly. Add 2 μL 6X sample loading buffer (obtain this from TA) to each reaction. Mix by pipetting gently, being careful not to introduce bubbles.

1	3
0.1	0.3
0.4	1.2
8	24
0.5	X
10	30

Electrophoresis

retrieve the horizontal gel apparatus from the common drawer of your station.

Hang the 10 well comb in the upper slot of the running tray, such that the thick teeth are down.

Prepare 40 mL of 1% agarose by weighing out 0.40 g of agarose in a clean weighboat.

Make 500 mL 1X TAE by diluting 50X TAE buffer (2.0 M Tris acetate, pH 8.0, 50 mM EDTA) with ddH₂O. Mix. Measure out 40 mL of 1X buffer into a 125 mL flask, and add the agarose measured out above. Save the rest of the buffer.

Gently swirl the flask to disperse the agarose in the buffer and cover with plastic wrap. Heat 1 min in a microwave oven, until just boiling.

Carefully the pour cooled agarose solution into the running tray on the gel apparatus. Make sure that the comb is perpendicular to the edge of the gel and perpendicular to the bottom of the tray. Avoid air bubbles, especially around the teeth of the comb – if you see bubbles, gently pop them using a pipet tip. Allow the gel to cool completely

Load 12 μL of each restriction digest into the other wells. You only get one shot at this with your samples, so you may want to practice loading with a 12 μL sample of just loading buffer and ddH₂O. It is a good idea to start with the wild type controls, and then load each clone in order.

120V 400A 30mins

Ladder

WT-

WT+

M-

M+

Experiment 3

Introduction:

Analyze the results of the restriction digest and mutagenesis performed on plasmid PJR1U. Data from the gel electrophoresis combined with the results of the sequencing should be enough to prove whether the mutation was performed successfully.

( This week’s experiment aims to determine whether the mutation we desired to introduce into our plasmid and undesired mutations are present. Our plasmid DNA sequence will be analyzed using GENEWIZ and NCBI Blast.)

Procedures: To view and correct your own sequences, download and install a chromatogram viewer. Chromas Liteis a good program for Windows and is installed on the lab computers. 4Peaks is a good program for OS X.

• Install 4Peaks
• Upload GENEWIZ file (filename.ab1) showing the observed DNA sequence.
• Check for ambiguous sites in the sequence where “N” is the base, 
• ensure that all ambiguous sites are correctly called and assigned if possible.
• Guage the quality of the sequence
• A really good read will result in usable sequence information for 750-900 bp, but many are in the 400-500 bp range.
• Poor sequencing is evidenced by low intensity or multiple peaks throughout the entire chromatogram or by a shortened read

Lab this week is in silico.

Methods:

Analysis of the experimental sequence

Examine the chromatogram using chromas lite. 

Delete the first few nucleotides that have significant signal noise. 

Trim the end of the sequence as well where the signal makes reading the nucleotides unreliable. 

Fill in the Ns with the most likely nucleotide 

Save the file as a FASTA File

Blast Compariston

Perform a nucleotide blast of the mutated AP along with the normal AP from E coli. 

Analyze the region where the mutation should have occured 

determine whether the sequence change is present

Perform a protein blast to determine if the change occured at the proper location.

ExPASy Proteonomics

“ProtParam” – this provides a large range of information about the entered protein sequence, including theoretical pI, theoretical extinction coefficient (which will be useful in determining protein concentration after dialysis later in the semester), amino acid concentration, molecular weight, etc. Numerous extinction coefficients are provided; choose the correct value for your enzyme based on what you know about the structure of alkaline phosphatase.

Expasy K328A.pdf

AP Sequence

MKQSTIALAL LPLLFTPVTK ARTPEMPVLE NRAAQGDITA PGGARRLTGD QTAALRDSLS

DKPAKNIILL IGDGMGDSEI TAARNYAEGA GGFFKGIDAL PLTGQYTHYA LNKKTGKPDY

VTDSAASATA WSTGVKTYNG ALGVDIHEKD HPTILEMAKA AGLATGNVST AELQDATPAA

LVAHVTSRKC YGPSATSEKC PGNALEKGGK GSITEQLLNA RADVTLGGGA KTFAETATAG

EWQGKTLREQ AQARGYQLVS DAASLNSVTE ANQQKPLLGL FADGNMPVRW LGPKATYHGN

IDKPAVTCTP NPQRNDSVPT LAQMTDKAIE LLSKNEKGFF LQVEGASIDK QDHAANPCGQ

IGETVDLDEA VQRALEFAKK EGNTLVIVTA DHAHASQIVA PDTKAPGLTQ ALNTKDGAVM

VMSYGNSEED SQEHTGSQLR IAAYGPHAAN VVGLTDQTDL FYTMKAALGL K

 Experiment 4

Introduction

This laboratory aims to isolate the mutated alkaline phosphatase protein from E. coli. The results of this purification will be analyzed over the next several lab periods.

Materials

• JW 0374 cells transformed with the mutated pJR-1U DNA will be used to prepare the altered alkaline phosphatase protein
• the chromosomal copy of the phoA gene in JW 0374 cells has been damaged by the insertion of the KanR cassette
• their phosphatase activity must arise from expression of the phoA gene located on the plasmid.

Procedure:

Cell Growth

TAs will start a culture a day before lab with 5mL LB media and 100ug/mL ampicillin that will be inoculated with a single colony of the mutated JW 0374 E. coli.

The culture will be allowed to grow overnight at 37^oC

50mL of sterile LB with 50ug/mL will be inoculated with the 5mL culture and incubated at 37^oC for 5 hours.

1mL of the culture will be measured in the spectrophotometer at 600nm with LB media as the blank

We are looking for an absorbance between 0.3-0.7

T1	0.491
T2*	0.52

Lysis 

Place the culture flask on ice, transfer 0.5mL to a microfuge tube, and store the microfuge tube on ice

Obtain and weigh a 50mL centrifuge tube and transfer the cells

Centrifuge at 4^oC at 4800xg for 15min

Remove the supernatant and weigh the tube with the cell pellet

Calculate the mass of the cells

T1
T2*

Resuspend the pellet in 20mL of cold 10mM Tris-HCl, pH 8

Directing a stream of buffer on the pellet with a Pasteur pipette

It is easier to resuspend in 5mL and then add the remaining 15mL 

Uniformly suspend the cells

Spin at 4^oC at 4800xg for 15min

Discard the supernatant

Use a kimwipe to remove excess moisture from the tube while holding it upside-down

Add 4mL cold 40% sucrose with 0.1mM EDTA to the tube and resuspend

Incubate for 5min on ice and spin at 4^oC at 4800xg for 15min

Remove the supernatant

Add 4mL of cold ddH₂O, resuspend and incubate for 5min on ice

Add 80uL of 0,5M Tris, 5mM MgCl₂, 5mM ZnSO₄ and spin at 4^oC at 48–xg for 25min

Obtain a 15mL disposable tube, 10mL syringe, and 0.45um filter unit

Unwrap syringe, remove plunger, and place on kimwipe

Screw the filter on the syringe

Transfer the supernatant into the syringe and filter the supernatant into the clean tube

This is the crude lysate

Transfer 250uL of the crude lysate to a microfuge tube (store at 4^oC), and 30uL to another microfuge tube (store at -20^oC)

Discard cell pellets in liquid cell waste container, plastic materials in biohazard bag.

Note: (Keep cells on ice during following) Using 2 cultures, double everything

DEAE Column

Obtain a ring stand with clamp, disposable BioRad column, 50mL beaker to catch eluant, 4mL 50% slurry of DEAE sepharose anion exchange resin (equilibrated in 10mM Tris-HCl, pH 8), 5mL 60mM NaCl (10mM Tris-HCl, pH 8), 6mL 120mL 120mM NaCl (10mM Tris-HCl, pH 8)

Attach a plastic stopcock to the column, clamp the column on the ring stand, and place a beaker under the column

Rinse the column with 10mM Tris-HCl pH 8.

Using a Pasteur Pipette, gently add resin to the column so that the resin reaches the top of the narrow part of the column.

Open the stopcock and leave the resin to drain, avoiding air bubbles and drying out of the resin.

Wash again with 10mL of Tris-HCl pH 8, and allow to drain. As the buffer flows out, use a pH paper to make sure the pH of the elutant is 8

Set up two 15mL tubes labeling one “FT” and the other “wash” and 12-1.5mL tubes numbering them 1-12.

Load the crude lysate carefully on the bed and allow it to flow through and collect in a tube labeled “FT”

When the sample has run into the column, attach 2mL of 10mM Tris-HCL, and collect elutant in a tube labeled “Wash”

Wash the column again with 5mL of 60mM NaCl and collect 1mL fractions in tubes 1-5

Finally wash the column with 6mL of 120mM NaCl and collect 1mL fractions in tubes 6-12

Store all tubes at 4 degrees Celsius.

Experiment 5

Introduction:

This experiment aims to analyze the different elution fractions from the DEAE column ( from the previous experiment) by using the Lowry method to quantify total protein and enzyme activity assay to quantify the amount of AP-specific activity. The principle behind this experiment is to quantify how well the purification worked for the fractions of phosphatase activity and total protein content.

Part 1: Activity Assay:

• Make 20mL of 0.5mM pNPP assay buffer by mixing 1.5mL of 10mM pNPP and 28.5mL of AP assay buffer 
• Place 1000uL of AP assay buffer in the reference cell and 995uL of assay buffer in the sample cell
• Add 5uL of the crude lysate and take a 90-second time course at a wavelength of 400nm 
• Repeat this for the flow-through, the initial wash, and all 12 elution fractions – Note if there is no activity present, retry with 10uL or 20uL of the sample (If this fails as well, ask the TA for some WT AP to check that you are performing the assay correctly. If you see activity with the WT but not the mutant, consult with the TA for troubleshooting advice. You do NOT want to waste a lot of AP).
• Make a plot of the enzyme activity vs the fraction number and identify which fraction contains the most activity 
• Record which fractions contain activity, the activity of each fraction, and the volume used in the sample in a Table 
• Save 30uL of each fraction and store at -20 degrees C

	Crude Lysate	FT	W	1	2	3	4	5	6	7	8	9	10	11	12
A400 5ul	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
A400 10ul	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
A400 20ul	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Part 2: Lowry protein assay

Reagents:

30 mL Reagent A: 2% Na2CO3 in 0.1 M NaOH

0.3 mL Reagent B: 2% Sodium Potassium Tartrate

0.3 mL Reagent C: 1% CuSO4

Reagent D: 3mL of Phenol reagent with 0.6mL of 0.25mg/mL BSA

• Prepare reagent D by mixing 0.3mL of reagent B with reagent A. Mix and add 0.3mL of reagent C, mix again. This solution must be made fresh daily.
• Prepare the standard curve by adding the amounts of BSA stock (250 μg/mL) shown in the table on the right to a labeled set of 1.6 mL microfuge tubes. Adjust the total volume of each sample to 200 μL using the indicated volume of ddH₂O.

• Add 1mL of Reagent D to each tube and mix well. Let it stand at room temperature for 10 minutes. (This is the most critical incubation for consistent and reliable results. Each sample needs to be incubated forEXACTLY 10 min, not more, not less)
• Stagger the incubation times start one every 20 or 30 seconds to make sure that each tube is incubated for EXACTLY 10 minutes
• Add 100uL of Folin solution to each tube mix immediately, and incubate exactly 30 min at room temperature.
• Read the absorbance at 750nm for each sample;do this twice for the standard samples. Use the same cuvette for all samples. Blank the spectrophotometer using a sample that contains no protein.
• Plot absorbance vs concentration known samples. Fit the line using Excel. This is your standard curve.
• Prepare assays in the same manner as above for each unknown sample, using 25 μL of sample for each assay. Unknowns should include the crude lysate, the FT, the wash, and each elution fraction from the DEAE column.
• Using the standard curve, determine the protein concentration of each unknown sample. 
• Graph the protein concentration vs. fraction number (putting your crude lysate, FT, and Wash before the elution fractions).

AP known conc	abs
0	0.001
2.5	0.07
7.5	0.133
12.5	0.202
20	0.222
100	0.189

sample	abs
CL	-0.003
ft	0.015
w	0.021
1	0.015
2	0.019
3	0.024
4	0.022
5	0.034
6	0.022
7	0.031
8	0.030
9	0.044
10	0.019
11	0.032
12	0.022

Experiment 6

Introduction:

This experiment analyzes the elution fractions samples obtained during purification by using electrophoresis. An SDS-PAGE will use to separate the samples based on molecular weight to allow visual confirmation of where the AP is located in the elutions. After that we will use Western blot.

Procedure:

Two gels will be run; One gel will be stained with Coomassie blue; the other will be transferred to a nylon membrane and probed with an antibody against alkaline phosphatase.

Pouring an SDS Polyacrylamide Gel

Obtain 2 precast mini-gels and 15-well sample combs

• The resolving gel have been pre-cast (10% acrylamide, 375mM Tris-HCl, pH 8.8, 0.1% SDS)

Place one precast mini gel sandwich on each side of the apparatus between the green clamps so that the bottom of the sandwich is between the two white teeth on the apparatus.

• Make sure that the spacer plate (the larger glass plate) is pointed away from the rest of the apparatus.

Once both gel sandwiches are in place, move the green clamps into place in a “closed” figuration

• If you have done this correctly, sealed resevior.
• Test this by squirting water into the main chamber.

Carefully insert a comb into each respective space at the top of each gel and ensure all teeth are between the gel plates. 

• LEAVE 0.5 cm BETWEEN THE COMB AND THE SHORT PLATE

Prepare the stacking gel solution by combining:

• 667 μL 30% acrylamide, 29:1 acrylamide:bisacrylamide
• 1.25 mL 0.5 M Tris-HCl, pH 6.8
• 2.98 mL dH₂O
• 50.0 μL 10% SDS
• 50.0 μL 10% Ammonium persulfate

Mix by swirling gently to avoid foaming 

Obtain a pasteur pipet and a small bottle of TEMED (add TEMED last)

• Once added to your solution, TEMED will accelerate the breakdown of ammonium persulfate and initiate the free radical polymerization of the acrylamide.

Add 5uL of TEMED to beaker and swirl to mix

Carefully draw up the solution into the pasteur pipet, 

• Avoiding bubbles as much as possible. 
• Gently place the pipet tip against the inside of the spacer plate of one gel (in between the teeth of the comb) 
• allow the solution to run down the glass into the gel form. 
• Fill the space about 3/4 of the way. 
• Do the same to the gel on the other side.

Add enough gel solution to bring the level up to the top of the short plate (the inner plate of the gel sandwich). 

Empty the remainder of the solution in your pipette back into the beaker, 

Allow the acrylamide solutions in the beaker and the gels to polymerize undisturbed.

Preparing Samples for Gel Electrophoresis

Obtain 2X sample buffer from the TA. Retrieve the samples of E. coli cells, crude lysate, flow-through, wash, and column fractions that were set aside from your purification of the mutant enzyme.

Do the following to prepare your whole cell sample for electrophoresis.

• Spin in microfuge for 1min
• Discard supernatant
• Add 50uL ddH₂O and resuspend
• Add 50uL of 2X buffer and mix
• Place in boiling water bath for 3-4min
• Spin in microfuge after cooling for 15s
• Store at -20^oC

For other samples:

• Transfer 20uL of each to labeled microfuge tubes and add 20uL of 2X sample buffer to each

Load a maximum of 13 samples to run on the gel

Running SDS-PAGE

Underline bottom of the wells with the comb still in place

Slide the comb out of one gel at a sink

• Do not dislodge the plates

Squirt water in between the plates

• Washes unpolymerized acrylamide out of the wells

Repeat with the other comb

Place the apparatus into the larger gel box, matching up the colors

Prepare 500mL tank buffer from 10X stock

Fill reservoir with buffer

• Make sure the level goes over the tops of the shorter inner plates

Pour the remaining buffer into the lower gel box

Buffer should rise to the :Two gels” line

Use a kimwipe to dry the top of the gel box and electrodes

Select which gel will be used for the Western blot and which will be the “Coomassie gel”

Line up samples in the following order: total protein, crude lysate, marker, flow through, wash, fractions

Use gel-loading tips to slowly pipet4uL of each sample into the appropriate well

• Avoid penetrating the gel
• Don’t pipet above the wells so that the samples leak between wells
• Avoid using end wells

Place the lid on the gel box, adjust voltage to 120V, and run until the bromophenol blue reaches the bottom

• Make sure to line up the electrodes

Turn off power supply and remove lide

Pour out running buffer

Bring the western blot gel to the TA, stain the other with Coomassie blue

Coomassie Blue Staining

Cover the bottom of a plastic tray with a shallow later of Coomassie stain

• Make sure you wear gloves

Gently pry the short plate of the sandwich with a razor blade and cut off the upper right corner

Peel the gel of the plate and place it in the tray

• Rock the tray to immerse the gel

Place the tray on the orbital shaker for 15-20min

Hold the gel in the tray and decant the stain solution back into the bottle

Add destain I, rinse tray, and discard

Submergethe gel in destain I and place on orbital shaker for 30min or until bands are becoming visible

Decant destain I into waste bottle

Submerge gel in destain II and place on shaker overnight

• Background staining should disappear and leave protein bands

Image gel with IBright imaging system and transfer to USB drive

Electrophoretic Transfer

The TA will assemple a transfer sandwich with a semi-dry blotting apparatus

• Consists of several blotter papers soaked in Tris-glycine buffer, nylon membrance, our gel, and more blotter paper

The sandwich is placed between 2 large electrodes and transfer of the proteins to the gel is performed by ppassing electric current horizontally

The layers are dissasembled after transfer and the membrane is placed in a blocking solution (4% nonfat dry milk) overnight

Pre-stained markers should be visible on the membrane

AP WT	CL	FT	W *
6	7	8	9

Get expert help for Experiment Lab Reports and many more. 24X7 help, plag-free solution. Order online now!