The Molecular Marker and the Unknown Fragments of DNA - Research Paper Example

Practical Write-up (7 practicals) 26/03 - Practical 4: Restriction enzyme digestion of plasmid DNA. Electrophoresis of PCR product, native and digested plasmid DNA Date: 25/03/2012 Experimental number: 3SBS573 2011-12 Aims and objectives To separate plasmid DNA by agarose gel electrophoresis Introduction Electrophoresis describes separation of a charged particle such as DNA or protein under the influence of an electric field. An appropriate buffer such as TBE is important while carrying out electrophoresis so as to maintain a constant ionization of the DNA molecules. In separation of DNA, target DNA is first digested with restriction enzymes to smaller fragments which are then filtered by the gel matrix based on their sizes. Digestion of the target plasmid DNA helps in facilitating the resolution of the fragments of interest in the gel. High molecular DNA such as the undigested plasmid may pose difficulties because of its size and may migrate poorly due to its large size which may not be accommodated by the pores in the gel matrix. Methods: i. Restriction enzyme digestions of plasmid DNA The volume of the plasmid prepared in the previous experiment was checked using the P20 Gilson pipette to ensure there was at least 20µl for the following steps. We prepared four sterile Eppendorf tubes and labeled as follow; Table 1: labeled Eppendorf tubes and their constituents A undigested DNA B Ndel digested plasmid C HindIII digested plasmid D Plasmid digested with both Ndel and HindIII Water 13µl 12 µl 12 µl 11 µl Plasmid 5 µl 5 µl 5 µl 5 µl Buffer 10x 2 µl B1 2 µl N1 2 µl B1 2 µl B1 Ndel 0 1 µl 0 1 µl Hind III 0 0 1 µl 1 µl Using the P20 pipette was used to transfer sterile water, plasmid to specific Eppendorf tubes as shown in table 1 above. A P2 pipettor was used to transfer restriction enzymes, 1µl of Ndel enzyme to tube B, 1 µl of HindIII and 1 µl of NDEL and 1 µl of HindIII enzyme into tube D. in all the tubes we ensured the final total final volume was 20 µl. After mixing the reagents thoroughly, they were incubated at 37° C for 30 min. ii. Gel electrophoresis A gel tank was made by sealing a tray with a masking tape and positioned it on the working bench. A comb was placed in the tray ready for making the wells. 0.5 g of agarose was used to prepare 1% w/v agarose gel mixture in a glass Scott bottle with addition of 50 ml TBE buffer. The agarose - TBE mixture was heated in a microwave for around 30 minutes to ensure mixing of the components. 1 µl gel Red was added after the bottle had cooled and the gel solution was swirled to mix and poured gently into the gel tray. The gel was left to set for about 30 minutes and the tape and the comb removed. The gel was placed appropriately into the running while ensuring that the sample wells were at the cathode. iii. Sample preparation for electrophoresis After the incubation period, 4µl of 6x concentrated DNA loading buffer (B2) into each of the tubes. The contents were mixed gently flicking the tube and tapping gently. This step was repeated while adding 5 µl B2 DNA loading buffer into the PCR tube. The samples ware loaded in the agarose gel as follows; Lane 1: All the content of the 1kbp ladder provided (~3 ml) Lane 2: 20 ml of undigested plasmid (tube A) Lane 3: 20 ml of NdeI digested plasmid (tube B) Lane 4: 20 ml of HindIII digested plasmid (tube C) Lane 5: 20 ml of NdeI and HindIII digested plasmid (tube D) Lane 6: 5 ml of PCR product Lane 7: All the content of the 100bp ladder provided (~3 ml) iv. Separation by electrophoresis, staining and taking of the image After connecting the electrodes to the PowerPack, electrophoresis was allowed to run at 140 V for 30 min. The gel was taken to the UV visualize where it was illuminated at 200 – 280 nm Results Figure 1: Gel image showing the DNA ladder and the migration of the sample plasmid DNA in the gel Lane Number Description 1 Kbp marker, 0.5, 1, 2, 3, 4, 5, 6 8 2 Uncut plasmid 3 Plasmid cut with NdeI 4 Plasmid cut with HindIII 5 Plasmid cut with both Nde1 and HindIII 6 PCR product of gene for red fluorescent protein 7 100 bp marker. Bands 100, 200, 300, 400, 500/517, 600, 700, 800, 900, 1000 Discussion The uncut plasmid DNA did not migrate considerably due to its large size as can be observed from the gel image above. However by digesting the DNA with the two restriction endonucleases short fragments of DNA which could penetrate the gel matrix were able to migrate and their sizes may help in determining the size of the initial plasmid DNA. PCR products as of the red fluorescent gene migrated considerably indicating their small sizes of the products. However as can be seen in lane 6 of the gel image they formed a smear which may present difficulties when ascertaining the sizes of DNA Conclusion In conclusion, agarose gel provides a matrix for separating DNA products and which help in determining sizes of DNA of interest. Short fragments migrate the farthest due to their small sizes towards the positive anode end of the gel. However large fragments suffer a higher frictional force with the gel matrix reducing their migration capability and thus move short distances. The total size of our plasmid may be determined from the total individual sizes of fragments digested with restriction and then compared to the plasmid digested with both restriction enzymes to obtain a relative molecular weight of the plasmid DNA. The molecular marker used in the experiment is of resolves into fragments of known sizes and thereby it is used as the benchmark for determining the sizes of te unknown fragments of DNA such as for the plasmid DNA. References Title: Practical 5 - Title: isolation of recombinant red fluorescent protein (RFP) from bacteria by affinity chromatography Date: 25/03/2012 Experimental number: 3SBS573 2011-12 Aims and objectives Purification and analysis of RFP protein using affinity chromatography Introduction Methods The cell pellets (A) from Escherichia coli (approx. 0.15 ml) were resuspended in 630 µl lysis buffer (P1) and 70 µl lysozyme stock solution (P5) was added with a further addition of 5 µl endonucleases (P4) and the solution mixed and incubated in ice for 15 min. the cell lysate was centrifuged at 12,000 x g for 10 min at room temperature and the supernatant collected. 50 µl of the supernatant was transferred to a new Eppendorf tube and labelled TE (total extract). The spin-column was equilibrated by adding 600 µl P1 buffer and centrifuging for 2 min at 890 x g and the flow-through disposed. Gently we loaded 600 µl of the cell lysate prepared earlier into the spin-column and centrifuged at 270 x g for 5 min. the collected flow-through was labelled FT. the spin-column was washed with 600 µl P2 buffer by centrifugation and the first was fraction discarded. The washing was repeated and 100 µl of P3 buffer was used to elute the protein by centrifugation and the eluted fraction labelled E1 for “first eluate”. This step was repeated and the eluted fraction labelled E2 for second eluate”. Results Discussion Conclusion References Practical 6: Title: protein separation using sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) Date: 25/03/2012 Experimental number: 3SBS573 2011-12 Aims and objectives To Estimate purity of a given protein sample Introduction The SDS-PAGE is a popular method for analyzing proteins qualitatively thereby monitoring the purity of proteins. However this method relies on denaturing proteins by using agents such as mercaptoethanol which breaks the disulphide bonds and rendering all protein molecules present in the sample as negatively charged. Therefore the mobility of the protein in the gel matrix will now depend on the protein size and not the charge. Methods Polyacrylamide gel preparation In preparation of PAGE, two gels are prepared a stacking gel and a resolving gel. The resolving gel was poured first to around 3 cm from the top of the glass plate and allowed to polymerize before pouring the stacking gel on top. Buffers for the stacking and resolving gel were prepared as shown in the table below; Table 2: buffers for the stacking and resolving gel Stacking gel (4%) Resolving gel (10%) 30 % acrylamide / bisacrylamide 0.33 ml 1.66 ml Stacking gel buffer (0.5M Tris-HCl pH 6.8 ) 0.63 ml Resolving gel buffer (1M Tris- HCl p H 8.8 1.90 ml Deionised water 1.42 ml 1.39 ml 10% w/v SDS 25 ML 50 µL Appropriate volumes of ammonium persulphate and TEMED (N, N, N’,N’- Tetramethylethylenediamine were added to the beaker containing resolving gel components as shown below in the table and the mixture was swirled to ensure an even polymerisation. Stacking gel (4%) Resolving gel (10%) 15 % w/v ammonium persulphate (APS) 25µl 50µl TEMED 2.5µl 2.5µl The resolving gel mixture was transferred quickly between the plates ensuring it reached the mark at 3 cm from top of the plate. A dropper was used to transfer a few drops of water saturated with butan-1-ol on the top of resolving gel mixture. The resolving gel was left to polymerise for about 10 min and the water saturated with butan-1-ol poured. Stacking gel was prepared from the beaker containing the stacking gel buffer prepared earlier in the experiment. Appropriate amounts of ammonium persulphate and TEMED were added as shown in the table 3 above. A comb for making the loading well was fitted into the stacking gel and the gel left to polymerise. Molecular markers and protein sample preparation Samples prepared form previous experiment molecular markers were loaded into the gel in this step. 25µl of 2x concentrated sample buffer was mixed with an equal volume of the protein sample in an Eppendorf tube. Each of the five tubes was labelled (MW, TE, FT, E1 and E2) and placed in a rack in a boiling water bath for 2-3 min. Assembling and loading the gel The gel was carefully taken from the stand and positioned in the gel running tank. 10x SDS-PAGE running buffer were diluted with deionised water to a final 1x concentration. This buffer was used to fill the lower (anode +ve) and upper (cathode –ve) chambers while avoiding introduction of ‘air locks’. The specified volumes of the MW marker and 20 µl of each sample were loaded into separate wells. Electrophoresis, staining an image captures After fitting the lid apparatus, the electrodes were connected to the PowerPack and electrophoresis allowed to run at 110 V for 30-40 min or until the tracking dye (bromophenol blue) reached 1 cm from the end of the gel. The gel was stained using the Kurien and Schofield method (1998) and gently removed from the glass plates. We placed the gel in a plastic dish and added 20 m of Coomasie Brilliant Blue R-250 stain. The plastic dish was covered and placed in a microwave in the fume chamber and the liquid heated to boiling point. Coomasie stain waste was poured and the gel was destained by adding 20 ml of 10% v/v acetic acid and heated until boiling. A gel scanner was used to capture the gel image Results Discussion Conclusion References Kurien, B.T. and Schofield, R. H. (1998) Heat mediated quick Coomassie blue protein staining and destaining of SDS-PAGE gels. Indian Journal of Biochemistry and Biophysics , 35, 385-389 Practical 7 Title: SDS- PAGE and Western blotting for the identification of a single component in a complex mixture of proteins. Date: 25/03/2012 Experimental number: 3SBS573 2011-12 Aims and objectives Introduction Methods: Preparation of Western Blotting components As the gel was running in practical 6, we prepared the components of Western blotting. For each gel we took two fibre pads, two pieces of 3MM Whatman filter paper and a piece of nitrocellulose membrane and placed them in a sandwich box. Approximately 50 ml of deionised water was added while ensuring the pads, filter and membrane were wetted out. The water was then disposed and 50 ml Western blotting buffer added and the components were left for 5 min in the buffer to equilibrate. Setting up the Western blot While wearing gloves, we prised apart the glass gel plates and placed the gel in the western blotting buffer solution in the sandwich box for 2 minutes. The Western blotting ‘sandwich’ was assembled on a level part of the bench. The transfer of the gel should be done quickly to ensure the components do not dry up. The sandwich was transferred to the apparatus of western blotting. Sandwich should be prepared while ensuring the gel is adjacent to the black part of the cassette. A stirrer was placed in the tank and the entire apparatus then placed on a stirrer block. A cooling pack was placed into the apparatus and the chamber was topped up with Western blotting transfer buffer making sure the gel and blotting papers were fully submerged before connecting the electrodes. After a 30 min transfer period of the Western blot at 100 V the Power Pack was turned off and unplugged from the socket. The cassette was opened and gently the different layers were peeled off. We marked the left hand corner of the side of the nitrocellulose membrane while it was intact with the gel. Staining with Ponceau Red dye The nitrocellulose membrane was lifted from the gel using tweezers and placed face-up in a lunch-box and then covered with Ponceau S solution. This was allowed to stand for a minute and then the dye was decanted back into a container. Approximately 20 ml of deionised water was added and rinsing done carefully until the MW and sample bands were visible. Again using tweezers the nitrocellulose membrane was lifted from the lunch-box and placed on an absorbent paper. The membrane was then cut into four strips where each strip contained the separated MW markers and proteins in the TE sample prepared in practical 3. Membrane blocking and immunostaining The membrane was placed in a tube while the face of the membrane was positioned to face towards the inside of the tube and the largest marker at the top of the tube. 10 ml of bovine serum albumin blocking solution (BSA) was added and the tube placed in a rocking platform for 15 min. the blocking solution was the poured off and washed away by adding TBS-T to the mark half way up the tube and then shaken by hand for 10 min. This washing step was repeated.5 ml of alkaline phosphatase labelled-mouse anti His-tag antibody solution was added and incubated on the rocking platform for 15 min and then the antibody solution was poured off. The membrane was then rinsed four times using TBS solution. Colorimetric detection using BCIP/NBT 10ml of BCIP/NBT solution was made by adding 240 µl of both substrates solutions to the 10 ml TBS solution and a cap placed and contents mixed. The blot was then transferred to the sandwich box and placed face-up and BCIP/NBT solution poured on top. Observation of the appearance of the His tagged bands as the blue colour developed were made. After the bands had been visualized the BCIP/NBT was poured into a waste container and blot rinsed 3 times with deionised water. Results Discussion Conclusion References Practical 8 Enzyme-linked immune-sorbent assay (ELISA) for detection of red fluorescent protein. Title date and experimental Aims and objectives Introduction Methods Preparing references standards A series of dilution each 1 to 10 were made from the 10 µg/ml stock solution of RFP by taking 0.1 ml of the stock solution and transferring to a clean plastic tube. 0.9 ml of phosphate buffer saline (PBS) diluents was added and the contents were then mixed using vortex mixer. A 0.1 µg/ml standard was prepared by taking a tube of 1 µg/ml of RFP and 0.01 µg/ml standards was prepared by taking a tube of 0.1 µg/ml RFP. The diagram below is an illustration of the dilution procedure. Loading the ELISA plate 100µl of the RFP standards wee pipetted into the ELISA plate wells as follows 10 µg/ml RFP: A1, A2, A3 1 µg/ml RFP: B1, B2, B3 0.1 µg/ml RFP: C1, C2, C3 0.01 µg/ml RFP:D1, D2,D3 A blank was also loaded in E1, E2, and E3 by pipetting 100 µl of the appropriate buffer. The unknown test sample was loaded in F1, F2, F3 wells and 100 µl of pre-dilute anti-AP (labelled with alkaline phosphatase, AP) antibody was added and the solution mixed gently. The plate was then placed in a rocking platform at room temperature and incubated for an hour. After incubation, the plate was inverted into the sink and pated firmly onto a wad of tissue Washing ELISA plate 200 µl of PBS-C a was added to each well that was in use and incubated at room temperature for 2 min. the plate was emptied by flicking the liquid into the sink and then washed again four times. Colorimetric detection and data taking 100 µl of prepared the diluted substrate p- Nitrophenyl phosphate (pNP) was transferred into each well which was used and the plate incubated at 37°C for 30 min until a soluble yellow end product was observed. A plate reader was used to measure the absorbance at 405 nm. Results Table 3: ELISA results 1 2 3 A 0.130 0.129 0.140 B 0.162 0.172 0.166 C 0.201 0.198 0.204 D 0.233 0.244 0.227 E 0.242 0.245 0.262 F 0.153 0.163 0.162 G 0.023 0.021 0.022 Discussion Conclusion References Read More