Isolation of splenocytes Method: Trypan blue dye exclusion test Reagents required: Method:

Transcription

1 Isolation of splenocytes Method: a) Mice were sacrificed and spleens were dissected out and washed in chilled PBS. b) With the help of clean forceps, spleen tissue was teased and then mashed between frosted glass slides. The slides were washed with PBS. c) Single cell suspension was prepared by passing the suspension through sterile mesh. d) The splenocytes collected were then washed twice with PBS in a 1:1 ratio followed by a wash with RPMI medium and RPMI containing 10 % FCS. The cells were then counted by hemocytometer. Trypan blue dye exclusion test Reagents required: 1. Trypan Blue solution (0.4%) 2. Hemacytometer and microscope Method: a) The cell suspension to be assayed was diluted 1:1 with 0.4% trypan blue solution. b) Counting chambers of a hemocytometer was loaded with the diluted suspension. c) The number of stained cells and total number of cells was determined. d) Unstained cell count was taken as a measure of viable cells. Enzyme Linked Immunosorbent Assay (ELISA) Materials / reagents required: 1. ELISA plate (96 wells) / strips (Maxisorp, Nunc TM Immunomodule, Denmark) 2. Antigen (Protein sample) 3. Carbonate-bicarbonate buffer, ph 9.6; 100 ml: Na 2 CO g NaHCO g 4. Phosphate Buffered Saline (PBS), ph 7.4: NaH 2 PO g Na 2 HPO g NaCl g MilliQ water - 1 liter PBST: PBS containing 0.05 % Tween

2 5. Blocking solution: 3% bovine serum albumin or skim milk in PBS 6. Developing (substrate) solution: Citric acid g/ 20 ml; Na 2 HPO g/ 10 ml 3 ml of citric acid solution + 1 ml of Na 2 HPO ml water 8 ml of above solution + 8 mg ortho-phenylenediamine (Sigma) + 8 µl hydrogen peroxide solution (30%). 7. Stop solution : 5 N H 2 SO 4 Bicinchoninic Acid (BCA) Protein Assay: Sensitivity: 0.2 µg/ml to 25 µg/ml. Reagents required: 1. 1 mg/ml BSA solution 2. Reagent A,1 litre, ph : Sodium bicinchoninate g Sodium Carbonate (Na 2 CO 3.5H 2 O) g Disodium Tartarate (Na 2 C 7 H 4 O 6.2H 2 O) g Sodium Hydroxide (NaOH) g Sodium Bicarbonate (NaHCO 3 ) g 3. Reagent B: 0.4 gm cupric sulfate (5 x hydrated) in 10 ml distilled water. 4. Standard Working Solution: 49 x Reagent A + 1x Reagent B Method: a) Optimal dilutions of sample and standard (BSA) were made in a volume of 250 µl in a microtiter plate. b) Hundred microlitres of standard working solution was added and allowed to stand for 30 minutes at 37ºC. c) The absorbance was measured at 562 nm and protein concentration in sample was calibrated from absorbance vs. concentration curve for standards. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Reagents required: 1. 30% acrylamide, 100ml: Acrylamide g Bis-acrylamide - 0.9g M Tris HCl, ph

3 M Tris HCl, ph % Sodium dodecyl sulfate (SDS) in H % Ammonium persulfate in H Sample buffer, 2X : 5% Tris HCl (1.25M, ph 6.8) 2% SDS 5% 2-Mercaptoethanol 0.01% Bromophenol blue 11.6% Glycerol 7. Electrode buffer : 25 mm Tris 192 mm Glycine 0.1% SDS 8. Destaining solution : Methanol - 45% Acetic acid- 10 % Distilled H % 9. Staining solution: 0.1% Coomassie brilliant blue (CBB R-250) in destaining solution Electrophoresis conditions: 100V constant voltage. Recipe for one SDS gel: Solutions Resolving gel (12%) Stacking gel (5%) 30% Acrylamide 3.0 ml 400 µl Distilled water 2.9 ml 1.8 ml M Tris HCl, ph ml M Tris HCl, ph µl 10% SDS 75 µl 25 µl 10 % Ammonium persulfate 25 µl 9 µl TEMED 3.5 µl 2.5 µl Zymography Reagents required: % isopropanol in 10 mm Tris-HCl, ph mm Tris-HCl, ph % gelatin 208

4 4. Staining solution: 0.1 % CBB (as described for SDS-PAGE) 5. Destaining solution: as described for SDS-PAGE 6. Loading dye: 0.5 ml Tris ph 6.8, 0.2 g SDS, 1.16 ml glycerol and 1 mg bromophenol blue made upto to a total volume of 10 ml with distilled water. Method: a) Protein sample and loading dye (1:1) was run on a 12 % SDS-PAGE containing 10 % gelatin. b) Gel was washed with 25 % isopropanol in 10 mm Tris-HCl, ph 7.9 followed by distilled water and incubated overnight at 37 C in 100 mm Tris-HCl, ph 7.9. c) Gel was then stained with CBB and destained to observe a clear band of proteolytic activity. Western blot Materials / reagents required: 1. Nitrocellulose (NC) membrane 2. Transfer buffer, 1X; 1 litre, ph - 8.4: Tris base- 3.3 g Glycine g Methanol- 25 % 3. PBS and PBST (PBS containing 0.05 % Tween-20): As described for ELISA 4. Blocking solution: 3% skim milk in PBS 5. Developing solution, 50 ml: Sodium acetate- 50 mm, ph 5.0 Diaminobenzidine- 15 mg Hydrogen peroxide- 15 µl 6. Stop solution: Distilled water Bacterial media 1. Luria-bertani broth: Dissolved 10 g bacto-tryptone, 5 g yeast extract and 10 g NaCl in 950 ml DW. Adjusted the ph to 7.2 with 10 N NaOH and made the volume to 1000 ml. 2. LB Agar: Prepared 1000 ml of LB broth and added 15 g of bacto-agar and autoclaved. 209

5 3. LB-ampicillin agar: Prepared and autoclaved 1000 ml LB agar. Cooled to 55 0 C and added 1 ml of 100 mg/ml ampicillin (100 µg/ml) and poured into petri dishes (~ 25 ml/90 mm plate).all the media were autoclaved at 15 lbs/sq. at 1200C for 20 min. Reagents for plasmid isolation 1. TEG (Tris-HCl ph 8.0, 25 mm, EDTA, ph 8.0, 10 mm, Glucose, 1%): Dissolved ml of 1M Tris-HCL, ph 8.0, 0.1 ml of 0.5 M EDTA and 0.05 g glucose in 5.0 ml autoclaved DW. 2. 1% NaOH and 0.2 % SDS: Mixed 0.3 ml 10 N NaOH, 1.5 ml 10% w/v SDS and 13.2 ml autoclaved DW (fresh) M Tris-HCl, ph 8.0: Dissolved g Tris in 800 ml DW. Adjusted the ph to 8.0 with HCl (11 N) and made up the volume to 1000 ml N NaOH: Dissolved 8.0 g of NaOH pellets in 50 ml autoclaved DW % Ethanol: To 125 ml DW, added 375 ml distilled ethanol M Ammonium acetate: Added 28.9 g of ammonium acetate in 50 ml of autoclaved DW M EDTA, ph 8.0: Dissolved g of disodium ethylene diamine tetra acetate.2h 2 O to 400 ml DW. Adjusted the ph to 8.0 with NaOH pellets, made up the volume to 500 ml and autoclaved % SDS: Dissolved 10.0 g SDS in 90.0 ml autoclaved DW. Warmed and made the volume to 100 ml with autoclaved DW. 9. TE (10 mm Tris-HCl, ph 8.0, 1 mm EDTA ph 8.0): Mixed 494 ml DW with 5 ml of 1 M Tris-HCl, ph 8.0 and 1 ml of 0.5 M EDTA and autoclaved. Plasmid DNA isolation (Alkaline lysis method) 1. Inoculated single bacterial colony in 5ml LB medium overnight containing appropriate antibiotic, e.g. here ampicillin (50 µg/ml). The cells were pelleted down at 6000rpm, 15min 4 0 C. 210

6 2. The cells were thoroughly mixed with 150 l of TEG buffer by vortexing. Then cells were kept on ice for five-min. 3. Added 300µl of alkaline SDS was added. The solution becomes clear and slimy. 4. Added ice cold 200 l potassium acetate and incubated on ice for half an hour. This step precipitates all genomic DNA and cell debris. 5. Then centrifuged at 12000rpm for 30min,4 0 C. Then the clear supernatant was taken out and 0.6 volumes of isopropanol was added and kept on ice for min. Then centrifuged for 25min at 12000rpm, 4 0 C.The glassy pellet is difficult to see, thus care needs to be taken while rejecting the supernatant. 6. The pellet was given a wash with 70%ethanol and then with 100% ethanol. The pellet was air dried and dissolved in water and analyzed on 1%agarose gel. Preparation of competent cells: 1. Inoculated single colony of E.coli stain to be made competent, e.g. DH5α into a 5ml LB tube and grown overnight at 37 0 C. 2. The next day secondary culture was done (diluted 1ml in 100ml of culture) Grown at 37 0 C 250rpm, 2hrs approximately for the cells to reach OD of 0.3.OD more than 0.4 leads to decrease in competence i.e. decreases the efficiency of transformation. 3. The culture was aliquoted into prechilled polypropylene sterile tubes and left on ice. 4. The cells need to be kept on ice subsequently. Cells were pelleted down at 3000rpm,4 0 C for 7min.(higher speed affects the viability of cells). 5. The cell pellet was suspended gently in 5-6ml of ice cold CaCl 2 solution (see in reagents). Cells were pelleted down at 2500rpm,4 0 C, 5min. 6. Cells were resuspended in ice cold CaCl 2 and incubated on ice for min. Cells were pelleted down at same speed. 211

7 7. Cell pellet was resuspended in ice cold CaCl 2.This resuspension is final and needs to be done very well. The suspension should be kept on ice for about 1hr. 8. Finally the cells are aliquoted as 100 µl and stored at C. Any aliquot taken out should not be refrozen. Competence of cells is assessed by transformation with a known plasmid vector and seeing the number of colonies that appear.no. Of transformant colonies per aliquot (µl) X 10 5 ==No.of transformants per µg of DNA used for transformation. Competence of cells decreases with time. Restriction digestion Restriction enzyme buffer (10X) =(1X) final conc. DNA =1µg Restriction enzyme used =1U to completely digest 1µg of DNA. For complete digestion of DNA the 50µl reaction mix contains DNA appropriately diluted and 5µl of assay buffer. The volume is made up by good quality autoclaved water. Finally the enzyme is added. Incubation 37 0 C, 3 hrs. If the DNA has to be digested with two enzymes then it's important to inactivate the first enzyme. This is done by heating the reaction mix at 65 0 C for 15 min. The DNA needs to be precipitated which is done by adding 0.6volumes of ammonium acetate and 2.5volumes of 100% ethanol. Incubate at C, overnight. Spin down and wash the pellet with 75% ethanol. The pellet is air-dried and then reaction is put up with second enzyme. Ligation Buffer: 10µl Tris.HCl (1M) 10µl MgCl 2 (1M) 10µl DTT (100 mm) 70µl water 2.2 X Reaction Buffer (For Ligation) 20 mg PEG+100µl water+20µl ATP+40µl ligation buffer 212

8 For ligation, 5µl of 2.2X reaction buffer and various ratios of vector and insert.0.5µl of T4 DNA ligase is finally added. The reaction mix is incubated at 16 C, overnight. Transformation: For 100µl of competent cells ng of DNA usually suffices (in the volume of 10-20µl). DNA lesser than 1000ng can be used but better to dilute if more than that. 1. Competent cells with DNA were swirled gently and kept on ice for min. 2. This mix was then incubated at 42 0 C for 2 min and immediately put on ice and kept for 5 min. This treatment is called "heat shock treatment" which actually causes DNA to enter inside the cells. 3. The cells were revived with 300µl of LB media and kept at 37 0 C, 260rpm,1-2hrs. 4. The cells were plated on LB amp plates and plates kept for overnight incubation at 37 0 C. 5. Remaining part of transformation mixture can be stored at 4 0 C. Reagents for agarose gel electrophoresis 1. 6X DNA sample buffer: Dissolved 25 mg bromophenol blue and 3.0 ml glycerol in 10.0 ml autoclaved warm DW. Stored as 1.0 ml aliquots at -20 C 2. TAE (agarose gel electrophoresis): For 50 X stock added 24.2 g Tris base, 10 ml of 0.5 M EDTA and 5.7 ml of glacial acetic acid. in 100 ml DW. 213