• Learn operation of Electrophoresis tank and Electrophoreis Power supply.

• Read following documents

Electrophoresis is a comprehensive term that refers to the migration of charged solutes or particles of any size in a liquid medium under influence of an electrical field.

• Document and revise common laboratory informations related to the examinations

1. Whole blood sent in EDTA vacuttee is acceptable.
2. If required the procedure can be performed with clot of plain sample or flouride sample.
3. Perform the procedure with a batch of 5 to 10 samples.
   1. Complete analysis within 3 days of arrival of sample.
   2. Do not wash samples till day of analysis, because RBC gets nutrients from plasma. They are better preserved in whole blood.
4. If RBC have settled completely, remove supernatant plasma
5. If RBC have not settled completely, centrifuge 3000 rpm for 10 minutes before removing supernatant.

1. Prepare wash solution containing 10 mmol/L EDTA and 5% Glucose. Dextrose Wash Solution Preparation
2. -First wash
   1. Add dextrose- EDTA wash solution upto 4.0 ml in EDTA vacuttee.
   2. Allow thoroughly mixing of Dextrose-EDTA wash solution & cells parts of sample By inverting a few times.
   3. Centrifuge the EDTA vacuttee at 3000rpm for 10 minutes.
   4. Remove washing solution part from the vacuttee.
3. Second wash
   1. as above
4. -
5. Prepare a set of eppendroff cups with sample_id label to match the sample batch.
6. Take 50 ul of Hb A Hb F Hb S control(s).Take 50 microliter of control & tests samples in eppendroff cups.Add 50ul Haemolysate solution in each cups for hemolysis.Mix it properly.Make sure that sample becomes completely clear.

1. Slide should be thoroughly washed first with detergent powder.
2. clean it properly with tissue paper.
3. put it in NAOH solution for overnight.
4. Wash it again with running tap water and then with DI water.
5. Wrap it in tissue paper and put it in appropriate allocated box.
6. Before applying sample, put slide in NAOH wash solution for 10-15 mins.
7. Wash it, dry it and the apply samples.

1. Siliconization of Glassware and siliconized slide will be used as a slider.
2. After siliconization of glass slide, mark the slide with oil paint as identification mark.

1. Take two slides.( one is siliconized and other non-siliconized one)
2. Siloconised slide used as slider while non siliconized one use as gel holder.
3. Cut appropriate size X-Ray film like a strip without any cut, slightly smaller(around 2-3mm) then slide.
4. Take this X-Ray strip and stick it to non siliconized slide with feviquick at four angles and wherever you feel it needed to stick.
5. Place 2 slides on a levelled surface as shown in above figure.Cut a rectangle of slide size from a x-ray plate, cut a four boardered frame from a x-ray plate,so the thickness of gel is same as thickness of x-ray plate.

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2. 
3. prepare 1% Agarose with Glycine - Tris buffer
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5. while taking agarose powder in flask use funnel so power particles will not stick to glass surface of flask
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7. while taking buffer make sure it will not disturb agarose powder in flask,so slowly pour buffer from side.
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9. Then pour hot agarose gel on non siliconized slide carefully.Make a wedge of siliconized slide on non siliconized slide containing agarose gel(as shown in figure).
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11. Carefully press the non siliconized slide over the siliconized slide,so that air do not get trapped in between two slides.
12. Gel will be ready to use after 10-15minutes.
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14. Use only freshly prepared slide as slides kept in refrigerator cause significant increase in endosmosis.

Method 1

1. Bring used histopathology blade from histopathology department after permission of in-charge of that respective department.
2. Make 2mm slot at every 5mm distance to prepare comb.
3. Wrap 3 layers of cello tape at each end of applicator comb so that level of applicator comb will remain faction of a millimeter higher from the gel so can prevent through and through cutting of gel and spreading of samples beneath the gel.
4. 

Method 2

1. Make 2mm slot at every 5mm distance to prepare comb.
2. Rub lower sharper end on both side of slide(after last slot), make it slightly arond 0.1-0.2 mm shorter than full slide height. so applicator comb both side will remain faction of a millimeter higher from the gel and properly set on X-Ray film. that can prevent through and through cutting of gel and spreading of samples beneath the gel.
3. Make assembly as shown in photograph below for holding sample applicator over the gel.
4. 

1. Hold the prepared gel slide in your one hand.
2. With thumb of one hand,fix the x-ray film of non siliconized slide,slowly and carefully remove the siliconized slide by sliding it with other hand.
3. Take care that gel do not get broken down or wrinkled while removing the siliconized slide.
4. Loading sample over applicator:
   1. 
   2. Taking sample directly over applicator will result in loading of excess sample over gel.
   3. So first keep magnet attached applicator comb on a clean microscope glass slide to remove excessive sample application over gel.
   4. Take 2ul of sample with 2ul pipette from prepared sample cup and apply it over applicator.
   5. After slight elevation of applicator,Remove the microscope glass slide.
   6. Before application over non siliconized slide, take out applicator and remove excess sample from other side(opposite to sample sample application site) of applicator. so sample is present only on one side and it will prevent sample spreading. Now keep applicator on its place on assembly.
5. Loading sample over Gel:
   1. put prepared gel slide beneath the applicator.
   2. The applicator is put over one end of gel loaded slide about 1-1.5 cm away from margin.
   3. Wait for 3 minutes,allow the samples to get completely absorbed within gel otherwise samples will spread.
   4. Then remove the applicator carefully.
   5. 

1. Clean the electrophoresis tank with DI water and dry it with tissue paper before use.
2. Put the electrophoresis tank on a levelled surface.Fill the Glycine-Tris buffer (full saturated) in the two compartments(as shown in figure).
3. After successful sample application,put the gel slide in electrophoresis chamber over a microscopic glass slide.
4. Connect the gel with buffer through two strip of filter paper on each side. (take two filter paper strips and stick it together with buffer) & cover the chamber with its lid.
5. Connect the electrode on the side of slide containing sample well to the cathode (-) & other electrode to the anode of the power supply.
6. Set voltage at 500(maximum) and Set current at maximum & run for 15-20 minutes.After 15-20 minutes,switch off the power supply & remove the slides.

1. Put the sample slide in methanol for 10 minutes for removal of excessive water molecules around protiens.
2. Take away the slide from methanol solution and allow it to dry.
3. Do not touch the surface containing gel with samples.
4. With a piece of tissue paper,wipe away methanol from the opposite surface of the gel slide.
5. Denature protein and fix the gel by heating it in microwave for 1 minute.
6. Allow the slide to be cool.
7. Take around 2 ml of a staining solution Amido black stain on the slide and spread it with glass rod on slide so as to stain the proteins over the gel surface.
8. Remove the excess stain by putting in the destaining solution 5% glacial acetic acid for 5 minutes.
9. Allow the slides to dry at room temperature.

1. label the slide with appropriate sample id according to sample application.
2. Keep the labeled slide on RICHO-2000L2 for scanning as shown in below figure.
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4. Set the “Resolution” around “600dpi” as shown in figure.
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6. Set thr scanner as shown below
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8. Press on “start”.
9. Press on “#” for sending the file which is scanned.
10. Open the “file manager” in resident room computer.
11. Open the “File system”—–>Click on “Scan” folder—–>Do right click on the image which is scanned—–>Open wih “GNU Image Manipulation Programme”.
12. Slide will be seen as shown in below figure after clicking on GNU Image Manipulation Programme.
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14. 
15. Do right click on that selected area—–>click on “Edit”—–>Click on “Copy”—–>Do right click on that selected area—–>click on “Edit”—–>Click on “Paste as”—–>Click on “New image”.New image is seen as shown in below figure.
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17. 
18. After Clicking on “export” below figure appear.click on “Export” also.
19. 

1. Go to Application menu—–>Click on web browser—–>Open 10.207.3.240
2. Do Login from your login ID.
3. Click on “Edit”—–>Click on “Manage attachment” as shown in below figure.
4. 
5. 2 attachments should be done - 1st is “electrophorogram” and 2nd is “Interpretation” file.
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7. Do 2nd attatchment as shown in below figure.
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9. After click on “Browse”,follow the sequence from where you find the “interpretation file(txt file)”.
10. Application menu—→File manager—→Back up1—→Biochemistry—→Department—→Parameter details—→HBe—→hb interpretation—→select appropriate attatchment.
11. Click on “Edit” and enter relavant sample id as shown in below screenshot.
12. 

In Above mentioned image, left sided test tube showing purple colour which is prepared by adding 10 micro liter of blood sample in 1 ml of water. Right sided test tube showing red colour which is prepared by adding 10 micro liter of blood sample in 1 ml of Dithionate Buffer having dithionate powder. whenever sulfur content in dithionate powder gets detoriated it will not give such colour.

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1. Various types of hemoglobins
2. Hb A is moved fastest,then Hb F And Hb S moved very slowly, so its band is near application sites.
3. Correlate result of Hb electrophoresis with Dithionite test.

1. POUR AROUND 10 GRAMS OF SODIUM DITHIONIDE POWDER FROM THE MAIN BOTTLE TO A URINE CUP
2. CLOSE THE LID TIGHTLY , PARAFFIN IT AND STORE IT IN A DEEP FRIDGE
3. TAKE AROUND 50 EPPENDROF CUPS AND WEIGH EXACTLY 200 MILLI GRAMS AND POUR IT IN THOSE 50 EACH CUPS AND THEN PARAFFIN THEM
4. STORE THEM AT 2-8 DEGREE CENTRIGRATE AFTER LABELLING THEM

For Checking Quality of Dithionite powder always put blank(1 mL DI water with 10 uL wash RBC) with other sample and compare the colour. Purple color with dithionite test suggest good condition of powder. If Orange color appear then the quality of the dithionide powder is detoriated