summarize all the objectives

I attached 3 lab reports 
Write in the lab reports

1. Introduction
2. summarize all the objectives
3. short summaries the procedures
4. background
5. discussion 

– Lab 9 Preparation for Protein Purification

Objectives Grow transformed E. coli in preparation for isolation and separation of green fluorescent protein.

Backgrounds

When E. coli containing the pGLO plasmid is grown in a medium containing arabinose the green fluorescent protein (GFP) is expressed. GFP can then be Isolated and separated on a SDS PAGE gel.

Cultures will be grown at 32C as it is the optimum temperature for folding of the GFP.

Methods

Before the lab	· Use aseptic technique during set up and inoculation· Select 3 tubes of broth if using 2ml: Label two ara/amp and one amp. If using 5 ml tubes of LB broth use 2 tubes label one ara/amp and one amp.· Add (9ul /1ml) each of arabinose and ampicillin to two tubes (ara/amp)· Add (9ul/1ml) of ampicillin to the third tube (amp)· Thaw one of your frozen culture tubes and mix· Add 100ul of frozen cultures to each tube· Place tubes in a rack and incubate in a shaker overnight at 32oC. Be sure to shake vigorously.
During the lab	· WEAR SAFETY GLASSES – Use a UV lamp to view each tube – remark on your observations· Transfer 2ml of culture from an ara/amp tube to a 2ml microfuge tube and spin for 5 minutes· Remove the supernatant into a waste beaker by either pouring or using a pipette – be careful not to disturb the pellet.· Add 250ul of TE buffer to the tubes and mix in the vortex mixer until the pellet is suspended· Add 50ul of lysozyme to this tube and mix· The tube will be collected and placed in the freezer for at least 24hr to lyse the cells· Select the remaining ara/amp and amp tube. Label two microfuge tubes.· Transfer 600ul of the ara/amp tube to a microfuge tube and 300 ul of the amp tube to the another microfuge tube.· Spin for 5 minutes and discard the supernatant.· Freeze these tubes and their pelletsLAB 10 Topic: Protein Purification
Objective	· Isolate and separate green fluorescent protein

Background

· When E.coli containing the pGLO plasmid is grown in a medium containing arabinose the green fluorescent protein (GFP) is expressed. GFP can then be isolated and separated on a SDS PAGE gel.· The hydrophobic interaction column (HIC) allows the separation of hydrophobic proteins. GFP has several stretches of hydrophobic amino acids: this will enable it to stick to the beads in the HIC.·

Materials	Lab· Samples of lysed cells from lab 9· Eppendorf tubes (sterile)· Test tube racks· P-1000 (200-1000 ul) – sterile· P-200 (20 to 200 ul) – sterile· Microfuge· UV light· HIC column (Biorad)· Elution tubes· Binding buffer (4M NH4SO4/TE pH 8)· Equilibration buffer· Wash buffer· TE buffer (elution)_____________________________________________________________
Lab Procedure	· Wear safety glasses and gloves.· Remove the Eppendorf tube containing cells from Ara/amp broth and lysozyme from freezer.· Thaw on the benchtop.· Centrifuge the tubes for 10 minutes to bring down the bacterial debris.· While the tubes are spinning shake the column vigorously to suspend the beads, then shake the column down to pack the resin in the bottom of the column. Tapping the column on the table top will also help settle the beads to the bottom.· Remove the cap and snap off the bottom of the prefilled column. Allow it to drain for 3 to 5 minutes into a tube. Do not jam the column into the tube as it will make a seal preventing drainage. You can use a piece of folded paper between the tube and the column to prevent this.· Prepare the HIC column by adding 2ml of equilibrium buffer (1ml at a time).· Drain the column to the 1 ml mark and cap the top and bottom.· After spinning your tube transfer 250ul of the supernatant into a microfuge tube and label with the number of your colony.· Add 250ul of binding buffer to the microfuge tube containing the supernatant.· Label 3 collection tubes 1, 2, 3 and place in a rack.· Drain the column into a waste container – another tube will work .· When the buffer has reached the surface of the resin, place it on collection tube 1.· Carefully load 250ul of the supernatant on to the column by resting the pipette against the side of the column. Let the entire 250ul flow into the column.· You can observe what is happening with a UV light.· make sure you are wearing glasses.· Transfer the column to tube 2· Add 250ul of wash buffer and let it flow into the column· Observe what is happening with a UV light· Transfer the column to tube 3· Add 750ul of TE (elution buffer) and let it flow through the column. You can observe with a UV light· Seal the tubes with parafilm and store at 4oC

LAB 11. SDS – PAGE EXPRIMENT

Objective

· Separate protein on an SDS-PAGE gel.· Determine the purity of GFP obtained during protein purification.· Separate the proteins from whole cell extracts of cells grown in the presence of ampicillin only and others grown in the presence of ampicillin and arabinose.

Background

Sodium dodecyl sulfate (SDS) is an anionic detergent that denatures proteins by breaking non-covalent bonds forming a micelle that confers a negative charge proportional to the polypeptide length. This allows proteins to migrate by size alone.

Materials	· P-20 (2 to 20 ul)· P-200 (20 to 200 ul)· P-1000 (200-1000 ul)· Constar gel load tips· SDS PAGE Gel boxes Invitrogen· Pre poured SDS PAGE gels – 4 to 12% – two / group· NUPAGE LDS sample buffer (4x)· Samples from GFP purification· Lysate· Tube #1· Tube #3· Tube #3· Samples of whole cells· ara/amp· amp· Protein ladder – Biorad Precision Plus Protein Kaleidoscope standards· Box 9· Buffer MES SDS running buffer 20X· Razor blades to cut gel· Heating block at 95oC· Coomassie blue stain 1X – Carolina Labs 219784· Protein gel destainer 5X – Carolina Labs 219785_______________________________________________
Procedure	· Wear nitrile gloves to protect you from acrylamide and stain.Preparing running buffer· Make up the running buffer – add 50 ml of the MES SDS 20X buffer to 950 ml. Invert the cylinder several times to mix the buffer.Preparing gels· You will prepare 2 gels / box· Peel off the tape covering the slot on the back of the gel cassette.· Pull the comb out of the cassette in one fluid motion to expose the gel loading wells.· Use a pipette to gently wash the cassette wells with 1X running buffer.· Invert the gel and shake to remove buffer. Repeat twice.· Fill the sample wells with running buffer.· Note: Be sure to displace all air bubbles from the cassette wells as they will affect sample running.· Lower the Buffer Core into the Lower Buffer Chamber so that the negative electrode fits into the opening in the gold plate on the Lower Buffer Chamber as shown in the figure.· Insert the Gel Tension Wedge into the XCell the buffer core.· Make sure the Gel Tension Wedge is in its unlocked position allowing the wedge to slip easily into the XCell SureLock unit.· The Gel Tension Wedge should rest on the bottom of the Lower Buffer Chamber.· Insert gel cassettes into the lower buffer chamber.· Place one cassette behind the core and one cassette in front of the core.· For each cassette, the shorter “well” side of the cassette faces in towards the buffer core.· The slot on the back of the cassette must face out towards the lower buffer chamber (see figure below).· Pull forward on the Gel Tension Lever in a direction towards the front of the XCell SureLock unit until lever comes to a firm stop and the gels or gel/buffer dam appear snug against the buffer core (see figure below).· Fill the Upper Buffer Chamber with 200 mL of the appropriate running buffer.· Use enough running buffer to completely cover the sample wells.· Ensure that the Upper Buffer Chamber is not leaking.· If the level of running buffer drops, the electrophoresis core and cassettes are not properly seated.Now prepare your samples – once they are ready to load continue· Use the pipette equipped with a round sample loading tip to underlay the samples into the gel wells (see figure below).· Lower the tip to the bottom of the sample well and slowly pipet sample into well without contaminating another well with the sample.· Fill the Lower Buffer Chamber (anode) by pouring 600 mL of running buffer through the gap between the Gel Tension Wedge and the back of the Lower Buffer Chamber as shown in the figure below.· Align the lid on the Buffer Core. The lid can only be firmly seated if the (–) electrode is aligned over the banana plug on the right. If the lid is not properly seated, no power will go through the mini-cell.· With the power off, connect the electrode cords to power supply {red to (+) jack, black to (–) jack}.Caution: Power must be off before connecting the XCell SureLock Mini-Cell to the power supply.Sample preparationHIC Column Eluates for gel 1· In microfuge tubes prepare samples for loadingComponentsVolumeSample volume15ulNUPAGE LDS Sample buffer15ulTotal volume30ul· Prepare samples of each of the samples (tube 1,2,3 and lysate if you have it).· Heat all the samples at 95oC for 10minsCell Pellets· You will have two pellets from lab 9: ara and ara/amp· Add buffer to each tube as belowComponentsVolumePelletNUPAGE LDS Sample buffer200ulTotal volume200ul· Aliquot 20 ul of ara and ara/amp two separate tubes labeled ‘heat’· Aliquot 20 ul of ara and ara/amp two separate tubes labeled ‘no-heat’· Incubate the ‘heat’ samples for 10 minutes at 95oCSample Loading· Use the pipette equipped with a protein gel loading tip to underlay the samples into the gel wells (see figure below).· Slowly draw the sample up into the tip as it will take time to fill due to the narrow bore.· Lower the tip to the bottom of the sample well and slowly pipet sample into well without contaminating another well with the sample.Gel 1 – Samples for gel lanes· Protein ladder – 5ul· Tube #1 – 10ul· Tube #2 – 10 ul· Tube #3 – 10 ul· Lysate – 10 ul or leave blank· Protein ladder 5ul· Tube #1 – 15 ul· Tube#2 – 15ul· Tube #3 – 15 ul· Lysate – 15ul or leave blankGel 2 – samples lanes· Protein ladder – 5ul· amp – heat· amp- no heat· ara/amp- heat· ara/amp – no heat· protein ladder – 5ul· amp – heat· amp- no heat· ara/amp- heat· ara/amp – no heatRun the gel· Fill the Lower Buffer Chamber (anode) by pouring 600 mL of running buffer through the gap between the Gel Tension Wedge and the back of the Lower Buffer Chamber as shown in the figure below.· Align the lid on the Buffer Core. The lid can only be firmly seated if the (–) electrode is aligned over the banana plug on the right. If the lid is not properly seated, no power will go through the mini-cell.· With the power off, connect the electrode cords to power supply {red to (+) jack, black to (–) jack}.· Run at 250V until blue dye is close to the bottom of the gel – but not so it runs off the gel (about 30 minutes)At the end of the run· At the end of the run, turn off the power and disconnect the cables from the power supply. Remove the lid and unlock the Gel Tension Lever.· There is no need to remove the Gel Tension Wedge.· Remove the gel cassettes from the mini-cell. Handle gel cassettes by their edges only.· Lay the gel cassettes (well side up) on a flat surface, such as the benchtop. Allow one edge to hang ~1 cm over the side of the benchtop.· Carefully insert the Gel Knife’s beveled edge into the narrow gap between the two plates of the cassette.· Note: Do not push the knife forcefully between the cassette plates or you may cut into the gel.· Push up and down gently on the knife’s handle to separate the plates. You will hear a cracking sound which means you have broken the bonds which hold the plates together. Repeat until you have broken the bonds on one side.· Rotate the cassette and repeat Steps 5–6 of this procedure until the two plates are completely separated.· Upon opening the cassette, the gel may adhere to either side. Remove and discard the plate without the gel, allowing the gel to remain on the other plate.· If staining, remove the gel from the cassette plate by one of the following methods:· If the gel remains on the shorter (notched) plate, use the sharp edge of the Gel Knife to remove the bottom foot of the gel. Hold the Gel Knife at a 90° angle to the gel and the slotted cassette plate. Push straight down on the knife to cut the gel. Repeat the motion across the gel to cut the entire foot. Hold the cassette plate and gel over a container with the gel facing downward. Use the knife to carefully loosen one lower corner of the gel and allow the gel to peel away from the plate.Coomassie Blue Staining· Ready-made Coomassie blue – Carolina labs· 5X destain solution (Carolina labs) – dilute to 1X before use
Data Analysis	· Photograph the destained gel on an illuminated box each.· Create a standard curve of the protein standards plotting log of molecular weight against distance moved from the wells.· Using a standard curve of the protein standards calculate the size of the GFP purified in the:· Column purification samples· In the cell pellet lysates of the lb/ara/amp. You are only going to size the band(s) that is darker in the lb/ara/ amp sample.
Questions	Purification· What are the roles of the Equilibration, binding, wash, and elution buffers· Which buffer has the highest salt contentSDS- PAGE· Are there differences between proteins seen between the cell pellets from lb/amp and lb/ara/amp?· What is the size of that band that is different in the pellets· Does the size of the band(s) match the known molecular weight of GFP size?· What did you observe in the gel of column fractions? Where you able to see purification of the GFP?· What is the size of the band you purified using the column.

 

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