Lab Report PDF

NAME HABIBA SALAH ELDIN SAAD CODE 2228296 DAY /TIME THURSDAY (8:2) GROUP C LAB1 PLASMID MINI PREP METHODOLOGY: 1. Harvest bacterial cell cu...

NAME HABIBA SALAH ELDIN SAAD CODE 2228296 DAY /TIME THURSDAY (8:2) GROUP C LAB1 PLASMID MINI PREP METHODOLOGY: 1. Harvest bacterial cell culture ( 1.5 ml ) by centrifuge at 8000 rpm for 2 min. at room temp. 2. Discard the supernatant 3. Resuspend pellet in 100 ul sol.1 by vortexing or pipetting until no cell clumps 4. Add 150 ul sol.2 and mix by inverting 4:6 times for 5 min. until sol. Become clear (lysis) 5. Incubate in ice for 5 min. 6. Add 150 ul sol.3 and mix immediately by inverting 4:6 times (neutralization) 7. Incubate in ice for 5 min. 8. Centrifuge at 13000 rpm for 10 min. at 4 degree 9. Transfer 400 ul of supernatant to a new tube 10. Add 1 ml 100% isopropyl alcohol then mix by inversion (precipitation) 11. Incubate for 15 min. at _20 degrees 12. Centrifuge at 13000 rpm for 10 min. at 4 degree 13. Discard supernatant 14. Wash the pellet with 500 ul ice-cold 70% ethanol 15. Centrifuge at 13000 rpm for 5 min. at 4 degree 16. Discard supernatant then wait till dry 17. Dissolve pellet in 30 ul nuclease free water 18. Test 3ul on agarose gel 19. Store at _20 degree AGAROSE GEL PREPARATION: 0.5% agarose in 250 ml TAE bu^er SAMPLE PREPARATION: add 10ul sample + 2ul dye (load the 12ul in the well) Comment: no results are found after gel electrophoresis this may be due to 1. Taken bacteria didn’t contain plasmid 2. Bacterial plasmid was dead To solve this, doctors repeated the exp. again or change the bacteria to ensure presence of plasmid and that it is alive  LAB2 COMPETENT CELL AND TRANSFORMATION METHODOLOGY: (competent cell): 1. Pellet the cell by centrifuge at 4500 rpm for 5 min. (repeated 4 times bec. The pellet was loose) 2. Discard the supernatant and resuspend in 2ml ice-cold 0.1M CaCl2 ( 1/5 volume of original culture = 400ul) 3. Incubate in ice for 20 min. 4. Pellet cells by centrifuge for 15 min. at 4 degree 5. Discard supernatant 6. Resuspend in 80ul CaCl2 + 80ul of 40% glycerol ( 1/25 volume of original culture) 7. Take 50ul for transformation ( 110ul remained) (transformation): 1. Transfer taken 50ul to new vial then add 5ul ligation rxn. (1:100 ng DNA) and mix gently 2. Incubate in ice for 15min. 3. Heat shock in thermal block for 45 sec. then immediately transfer into ice for 1 min. 4. Add 250ul LB medium 5. Incubate at shaker incubator horizontally at 200 rpm for 45 min. 6. Transfer 50ul from transformed cells and spread on the LB-AGAR plate 7. Incubate 5 days Comment: blue and white colony appeared  LAB3 INDUCTION METHODLOGY: We used the 0.1 mM conc. From IPTG 1. Discard 1 ml from bacteria (noninduced) 2. Add 500 ul from IPTG on 5ml from bacteria (5.5ml) 3. Incubate for ½ h. 4. Take 1ml from falcon and reincubate for another ½ h. 5. Centrifuge the taken 1ml then discard supernatant 6. Hand on the pellet 7. Repeat step 4 and 5 at 2h. and 3h. Troubleshooting: if no pellet appeared after centrifugation this may be bec. 1. The taken culture wasn’t turbid so the bacterial concentration was low 2. Problem in IPTG COMMENT: best time for induction 1mM AND THE 3 h. LAB4 REINDUCTION AND SDS_PAGE Doctors reinducted the cells again at 1mM AND THE 3 h. Sample prep.: 1. Add 20ul sample bu^er (lammeali bu^er) to 20ul sample culture pellet and mix well 2. We did this steps for ( uninduced + 30 min. + 1h. + 2h.) 3. Heat in boiling water for 3:5min. (thermal block) 4. Chill in ice for 2:3 min. and centrifuge at max. speed for 2 min. to remove insoluble materials Troubleshoot: there was a problem in the conc. In the TEMED so we didn’t do a gel so samples are used in the next time ( WE CAN SOLVE THIS PROBLEM BY INCREASING TEMED CONCENTRATION)  LAB5 SDS_PAGE GEL PREPARATION: 1) Separating gel (12%) :. 1.5 M tris hcl (ph=8.8) 5ml. SDS 100ul. acrylamide 4 ml. dh2o 3.35ml. ABS 300ul. TEMED 20ul ( ABS and TEMED ADDED TOGETHER IN THE FINAL STEP FRESHLY PREPARED) 2) separating gel (4%) :.. 1 M tris hcl (ph=6.8) 2.5ml. SDS 100ul. acrylamide 2 ml. dh2o 5.3ml. ABS 200ul. TEMED 15ul ( ABS and TEMED ADDED TOGETHER IN THE FINAL STEP FRESHLY PREPARED) After gel preparation and wait till dry we will load from each sample ( prepared from last time 20ul) in every well and wait till migration COMMENT: no results are found on the gel Troubleshooting: 1. Bands in the stacking only: insu^icient voltage applied ( apply higher voltage) 2. No visible bands:. very low protein concentration (repeat the exp. ). improper loading of the sample. insu^icient staining or concentration of dye was very low (repeat exp. And change concentration of the dye)  LAB6 CELL EXTRACTION AND AFFINITY CHROMATOGRAPHY Cell extraction methodology: Sample prep for chromatography: 1. Sample washed from IPTG once with a large volume of binding bu^er 2. Resuspend sample in 20 ml binding bu^er 3. Sonification ( 6 times for 30 sec. with 30 sec. pause alternatively at 30 degree) 4. Add 20 ug/ml from DNase1 and 0.1 mg/ml lysozyme 5. Incubate in room temp for 45 min. 6. Centrifuge and supernatant will be used in purification A^inity methodology: 7. transfer 500ul NI_NTA resin in 10 ml plastic column 8. open the bottom 9. wash the resin 3 times (added 3+3+3+1) from (equilibration bu^er) overall 10 ml 10. load 5ml lysate 11. mix suspension gently 12. incubate lysate mixture on end_over shaker for 45 min. at 4 degree 13. remove bottom cap 14. collect the flow on two steps 15. wash twice with 4ml binding bu^er ( 2ml each time) 16. discard the flow 17. label 2 microcentrifuges for the ELUTE sample ( E1,E2) 18. elute protein 2 times with 0.5 ml elution bu^er 19. wash 2 times with 8ml binding bu^er (4ml each time) 20. wash the column one time with 8ml 30% ethanol 21. add 2ml 30% ethanol ( close the column at the top and bottom) store at fridge ( column must kept wet )  LAB7 BRADFORD PROTEIN ASSAY St. solution (18.70 ul) and 31.30 elution bu^er to make final volume 50ul This for 0.75 mg/ml 1. take 10ul from standard diluted sample and add 100ul Bradford on them (repeat this step two times ) then mix well and load directly in the well. (load Bradford first). (don’t change the pipette, make sure not to do any air bubbles while loading and mix gently after sample loading) 2. take 10ul from E1 twice after adding 100ul of bradford and load it. (make the same with E1 100ul ) 3. repeat step no.2 with E2 4. the first well contains Bradford and elution bu^er only (blank) 5. measure the absorbance of the standard protein using a spectrophotometer at 595nm 6. plot standard curve between concentration and absorbance 7. we can determine the conc. of the other measured samples of unknown protein concentrations from the linear equation of the standard curve comment: 1. E1 10UL + E2 10UL: the concentrations in this sample were out of the Bedford range ( 100: 1500 ug/ml) 2. E1100ul + E2100ul:those concentrations lies within Bradford sensitivity range