Cell Lysis
To the PBS were added protein inhibitors. Thereafter 200 ml pellet was resuspended in 1oml PBS with 100 μl lysozyme enzyme. The contents were then incubated at room temperature for 15 minutes on the rotary suspension mixer. Thereafter, the contents were freeze-thawed 7 times, first using liquid nitrogen, then in a water bath, at 37 oC. Then, 100 μl DNase enzymes were added, and the contents incubated on the rotary suspension mixer for 15 minutes. Spinning then followed at 12,000rpm for 30 min, at a speed of 4oC. The supernatant and the pellet were then placed in separate tubes and the sample loaded on SDS page.
HIS-Tagged Purification
Ni-NTA Equilibration
For complete mixing, the bottle was shaken well. 200 μl was taken from the bottle and the beads resuspended in the appropriate amount of a denaturing equilibration buffer. The contents were spined at speeds of 2000 rpm for 5 minutes, and the supernatant discarded. Step 3 was repeated to facilitate riding of any residual ethanol.
Purification of Soluble Proteins
The supernatant from the equilibrated beads was added and placed on a rotating mixer for 1 hour. Spinning was accomplished for 5 minutes at 700g, and the unbound supernatant discarded. The beads were then resuspended in denaturing buffer and spined for a further 5 minutes at 700g. The supernatant was discarded, but 100 μl of it was retained for SDS-PAGE- Wash 1. Thereafter, steps 5 and 6 were repeated. Again, the supernatant was discarded, and another 100 μl of retained for SDS-PAGE- Wash 2.
The beads were then resuspended in the same volumes of denaturing buffer, in addition to solubilising 20 mM imidazole as the beads. Spinning was accomplished for 5 minutes at 700g. The supernatant was discarded but 100 μl of it was retained for SDS-PAGE- IMIDAZOLE WASH. The beads were resuspended in a similar buffer volume as that of the beads, with 100mM imidazole, and spinning accomplished for 1 minute at 700g. All the supernatants was retained.
100 μl supernatant from the supernatant was taken for the SDS-PAGE (ELUTION 1). The beads were resuspended in with 250 mM imidazole in a similar volume of buffer to that of the beads used. Spinning was done for 1 minute at 700g. All the supernatant was retained, and a further 100 μl taken from the supernatant for SDS-PAGE (ELUTION 2). The beads were resuspended in the same volume of buffer with 1 M imidazole, and spinning accomplished for 5 minutes at 700g. All the supernatant was retained.
100 μl from the supernatant for SDS-PAGE was taken and a similar volume of buffer added back to the pelleted beads. The contents were then gently agitated for 1 minute. Boiling was then accomplished for 5 minutes at 100oC, followed by 5 minutes of spinning at 700g. All the supernatant was retained. 100 μl from the supernatant for SDS-PAGE was then taken and every sample ran on SDS page.
Agarose Gel Electrophoresis
The Agarose gel electrophoresis technique finds use in molecular biology and biochemistry for purposes of facilitating the separation of RNA and DNA fragments (Brody & Kern 2004) on the basis of their length (Kryndushkin et al 2003; Sambrook & Russel 2001). In addition, the technique is also useful in assessing the RNA and DNA size.
In preparing the gel, a flask was used to weigh agarose. To this was added 80 ml of 1x TAE. Then, the flask with its contents was placed in a microwave to facilitate thorough mixing for a period of 1 min. The contents were removed from the microwave and carefully mixed again, then placed on the bench and allowed to cool. Upon cooling, 3 μl of ethidium was added. As the gel cooled, masking tape was used to securely tape the two open ends of a gel boat. The desirable comb was placed in the boat, after which it was placed on the bench. 1x TAE was used to fill up the gel tank apparatus, making sure that the electrodes faced to the right. Upon the setting and solidification of the gel, the tape and the comb were both carefully removed and the gel placed in the tank. The tank was filled continuously until the level of the buffer had exceeded that of the gel.
During the process of loading and running the gel, 6x DNA loading dye was used to mix the DNA samples. The mixed DNA samples was then loaded into the wells
The lid was slid into the apparatus, and the black and red cords plugged into the powerpack. The gel was run for between 1 and 3 hours at approximately 80-110V. The Gel Doc System was used to visualize the gel.
Restriction Enzyme Digest
Restriction enzymes act at the restriction sites of single or double-stranded DNA (Pingoud, Alves & Geiger, 1993; Tovkach Zeevi & Tzfira 2010), and cut them at these sites.
To facilitate digestion, 10 µl of Dna was added. In addition, 2 µl of BSA was added. 2 µl of buffer was also added, and a further 1 µl retraction enzyme added. To this was added 5 µl Sterile Water, and all things were put in an epp tube.
The digestion process involved the addition of 10 µl of Dna, 2 µl of BSA, 2 µl of buffer, and 6 µl Sterile Water.Everything was then placed in an epp tube. The digest was run, and then taken on a gel agroass.
SDS-PAGE
The SDS-PAGE technique has found wide application in the fields of genetics, biochemistry, molecular biology, and forensic (Schägger & von Jagow 1987), among others. In this case, the technique is used to facilitate the separation of proteins on the basis of their electrophoretic mobility. In this case, the electrophoretic mobility of proteins depends on either their molecular weight, or polypeptide chain length.
The methodology of the SDS-PAGE technique involves obtaining a backing plate, glass plate, a comb, and 2 spacers. All the equipments obtained were thoroughly washed with the Pyroneg detergent, making sure that nay dirt from the plates was removed. Thereafter, ethanol was used to wipe down the plates, and they were let to dry. Caster stand, gel caster, and locks were obtained. The preparing solutions for resolving and stacking gels for Tris-Glycine SDS PAGE were also obtained. For the 12 % resolving, 3000 µL with a concentration of 40% Acrylamide was required. In addition, 2600 µL 1.5M Tris with a PH of 8.8, and 4192 µL H2O was also required. To this was added 100 µL 10% SDS and 100 µL 10% APS and 8 µL TEMED.
For the 5% Stacker, 3000 µL 40% Acrylamide was required. In addition, 4192 µL H2O and 2600 µL 1.5M Tris with a PH of 8.8 was necessary. In addition, 100 µL 10% SDS and 100 µL 10% APS and 8 µL TEMEDwould also be required. The apparatus was then added. After the gel polymerises had been resolved, the stacking gel was poured. The combs had to be placed into the stacking gel prior to its setting.
Tris-Glycine SDS-PAGE Gel Running
Once the gel had set, clips were used to suspend the gel on the electrophoresis apparatus. The SDS-Page Running Buffer was then poured in the space between the electrophoresis apparatus and the backing plate until the levels of the buffer matched that of the wells top. In addition, care was taken to ensure that the level of the buffer at the electrophoresis apparatus’ bottom had barely touched the gel’s bottom. Thereafter, the combs were carefully pulled out of the well. After that, SDS buffer was squirted into each well to facilitate washing. Samples were then loaded into the wells and 5µL Protein ladder added. The apparatus was then covered and the gel allowed running for between 1 and 1.5 hours at 20mA or until the gel had begun to run off.
Thereafter, the apparatus was dismantled, the gel placed in the lunch box containing the right solution, Pyroneg was then used to clean the plate equipment, and ethanol used to wipe it. All the items were then returned into the SDS-PAGE equipment box.
Samples preparation for the SDS-PAGE
Pellets were resuspended in dH2O or 1xPBS by utilizing the available OD values, in accordance with the relationship provided below:
O.D. = 100x buffer
100µL PBS= 1 O.D.
The most ideal amount of buffer was used to resuspend pellets, and vortex samples used to facilitate bacterial pellet break up. Samples were boiled for a period of approximately 5 minutes. Thereafter, the 6X SDS loading was added. Again the samples were boiled for a period of between 5 and 10 minutes. Approximately 20 µl samples were loaded onto the gel to facilitate the commencement of ++Q
Transformation
Transformation is a term used in reference to the ability of a cell to undergo through the prices of genetic alteration following uptake, integration as well as the expression of the exogenous DNA (Bruce et al 2002), often finding its way into the cell via cell membranes.
Procedure
A tube containing competent cells was placed on ice at -80oC up to the point of thawing. 2 µl of DNA was placed in a microfuge tube, and 50 µl of the competent cells added. The mixture was then allowed to incubate for a period of 15 min on ice. Thereafter, the contents were heat shocked for I min at 42°C. Again, the contents were incubate for I min on ice. 52 µl was taken from the tube and plated on LB plates using the suitable antibiotics. The plate was left in the incubator overnight, with the temperatures set at 37°C.
Sequencing
To the eppent tube were added one micro-liter of reverce and 11 micro-liter of DNA. In addition, another 11 micro-liter of DNA was added into an eppent tube and a further one micro-liter of forwarde. Thereafter, the contents in the pair eppent tubes were sent to check for sequencing
Plasmid DNA isolation
Eppi tubes of approximately 1.5 ml were used for holding overnight culture. The eppi tubes containing the overnight culture were then centrifuged for 5 minutes at a speed of 4000 rpm. The supernatant was removed and discarded and the pellet resuspended in a 100 µl homogenization buffer (cold solution I). The mixture was left to stand on the bench for a period of 5 minutes. 200µl lysis buffer (solution II) was then added. The content were inverted 5 times and again allowed to stand on the bench for a period of 5 minutes. Thereafter, 150 µl Solution III was added.
The contents were gently mixed and placed in the refrigerator for a period of 5 minutes. To this was added 350µl of 5M LiCl, followed by a through mixing. The contents were centrifuged for 5 min at 13,000 rpm. The supernanatant without any precipitate was drained into a new tube. To the supernatant was added 500µl cold isopropanol for purposes of facilitating plasmid DNA pelleting. The contents were centrifuged for 5 minutes at 13,000 rpm.
The supernatant was carefully removed, and 500µl ethanol with an alcohol concentration of 70% added. The contents were centrifuged for 5 minutes at 13,000 rpm. The supernatant was carefully removed the content was dried to facilitate ethanol evaporation, and then resuspended in 30µl TE. To this solution was added 3 µl of Rnase enzyme, and the sample was thereafter loaded on the gel
Mini Prep Kit
MiniprepsDNA Purification System
Approximately 1.5 ml of the overnight culture was placed in eppi tubes. The eppi tubes and the contents were centrifuged for 5 min at 10,000 × g. The supernatant was removed and discarded. The pellet was resuspended in a cell resuspension solution (250μl). 250μl Cell Lysis Solution was then added, and the contents mixed by inverting tubes 4 times, followed by incubation for 5 minutes. Alkaline protease solution (10μl) was added and mixing accomplished by inverting tubes 4 times, followed by incubation at room temperature for 5 minutes. 350μl Neutralization Solution was added and mixing accomplished by inverting tubes 4 times.
A maximum speed of 14,000 × g speed was used to centrifuge the bacterial lysate in microcentrifuge at room temperature, for 10 minutes. A decantation process was then used to transfer the cleared lysate to the already prepared spin column. The supernatant was transferred into a microcentrifuge and centrifuged at maximum speed at room temperature, for 1 minute. To the spin column was added 750μl Column Wash Solution. The content was placed in a microcentrifuge and centrifuged at room temperature for I minute at maximum speed. The spin column was then removed from the tube and the flow through discarded.
Afterwards, the spin column was reinserted into the collection tube. The wash procedure was repeated using 250μl of Column Wash Solution. The contents were transferred into a microcentrifuge and centrifuged at room temperature for 2 minutes, at maximum speed. The spin column was transferred to a new, sterile, microcentrifuge tube. The plasmid DNA was eluted by an addition to the spin column of 100μl of Nuclease-Free Water. The contents were transferred to a microcentrifuge and centrifuged at room temperature for 1 minute at maximum speed
Once the DNA had been eluted, the assembly was removed from the 1.5 ml microcentrifuge. The spin column was tubed and discarded. When stored at or below –20°C, DNA has the ability to remain stable in water without the need to add a buffer. The sample was then run on the gel agarroass
Western blotting
As an analytical technique, the western blot finds use in the detection of the availability of protein within a given tissue extract or homogenate sample (Hempelmann et al 1987; Towbin, Staehelin & Gordon 1979). In order to enhance the separation of the denatured or native proteins, the technique relies on the gel electrophoresis.
Transfer
The semi-dry transfer apparatus was first checked to ensure that it was dry. Afterwards, 3 sheets of blotting paper were cut to the exact size of the gel. The 3 sheets were wet in transfer buffer one-by-one. The wet sheets were laid down on the apparatus, one after the other, making sure to roll them flat. A strip of the membrane with a similar size to the gel was cut and then carefully removed from the blue backing paper. Care was taken to avoid touching the membrane, and little amount of buffer poured onto the blotting paper stack. The membrane was wet in the buffer after which it was carefully laid on the blotting stack and rolled. A little amount of buffer was added
The gel was removed from the gel plate cut of the stacking get, briefly wet and carefully placed on the membrane. The gel was then gently rolled flat, and steps 2-4 were repeated once more
Running the Apparatus
The gel/membrane transfer stack was dried to avoid any residual liquid. The lid was closed, and the contents run for 1 hr 15 min at mA = 150 for 1 hr 15 min. This facilitated the attainment of a transferring voltage of ~12V. When the voltage read 0, the transfer was stopped the lid opened, and the area around the stack dried once more. Once the transfer had been completed, the lid was gently lifted to check if the stack had not stuck to the top electrode. The blotting paper was then removed from the gel. The metallic surfaces of the two parts of the apparatus were rinsed in distilled water. The apparatus was then dried and reading taken for the next use.
Probing
The membrane was stained in Ponceau stain for 5 min, after which it was then destained for 5 min in distilled water. The membrane was then blocked with blotto and placed in the oven for I hour for incubation. This procedure serves to block all non-specific sites for protein binding. The membrane was washed 4 times using PBS-0.1% Tween, with each wash lasting 5 minutes. Primary antibody was a primary antibody was included and the contents incubated for between 1 hour and overnights on the basis of the primary antibody strength, and step 3 repeated.
A secondary antibody was included in the second incubation at room temperature, for 1 hour. Again, step 3 was repeated. Thereafter, all liquid was drained from the membrane, and the picture taken.
Protein Induction
Small scale
A single colony was taken and inoculated overnight into 2 ml LB/antibiotic overnight, in the shaking incubator at 37oC. The overnight culture from step 1 was added into 10 ml of fresh LB using the most appropriate antibiotic in a 50 ml falcon tube so that the new culture had an OD600 of 0.1. The contents were placed in the incubator at 37oC and shaken until the OD600 had reached 0.5-0.8. Once the correct OD600 had been attained, the value was recorded and 1 ml of culture removed, then placed in an eppendorf tube labeled IPTG.
The tube labeled IPTG was spinned for 5 min at 4000 rpm, and then the supernatant was drained. The pellet was stored in the freezer at -20°C to check on the gel. 1mM IPTG was added to the culture that had remained in the falcon tube every 1 hour, 1 ml of culture was then taken, the OD values recorded a new eppi placed, and steps 6-7 repeated once more. In addition, step 8 was repeated for 4 hours and the samples run on SDS-PAGE.
Large Scale
A single colony was taken and inoculated overnight into 15ml LB/antibiotic at 37oC. Enough of the overnight culture was added to a basted flask the next morning, and 400-500 ml of fresh LB/antibiotic added to attain an OD600 value of the new culture of 0. The contents were shaken for between 1 and 3 hours at 37oC until the OD600 had reached between 0.6 and 0.8.
Upon the attainment of the correct OD600 1 ml of culture was removed and placed in an eppendorf tube labeled IPTG. The IPTG labeled tube was centrifuged for 5 minutes at 4000rpm to facilitate the draining of the supernatant. The remaining content was then stored in a freezer at -20°C to check on the gel. 1mM IPTG was added to the culture that had remained in the falcon tube and shaking continued. The entire contents of the flask were poured into the tubes after 4 hours. The tubes with the contents were centrifuged for 20 minutes at 4000rpm. The supernatant was discarded and the lysis process of the pellet continued.
Reference List
Brody, J.R., & Kern, S.E., 2004. History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. Vol. 333, No. 1, pp.1-13.
Bruce, A. et al., 2002, Molecular Biology of the Cell. New York: Garland Science. Hempelmann, E., Schirmer, R. H., Fritsch, G., Hundt, E., & Gröschel-Stewart, U., 1987.
Studies on glutathione reductase and methemoglobin from human erythrocytes parasitized with Plasmodium falciparum. Mol Biochem Parasitol, Vol. 23, No. 1, pp. 19-24
Kryndushkin, D. S., Alexandrov, I. M., Ter-Avanesyan, M. D., & Kushnirov, V. V., 2003. Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104. Journal of Biological Chemistry, Vol. 278, No. 49636
Pingoud, A., Alves, J., & Geiger, R.,1993, Chapter 8: Restriction Enzymes. In Burrell, Michael. Enzymes of Molecular Biology. Methods of Molecular Biology. Totowa, NJ: Humana Press
Schägger, H., & von Jagow, G., 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem, Vol. 166, No. 2, pp. 368-379.
Sambrook, J., & Russel, D. W., 2001, Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Tovkach, A., Zeevi, V., & Tzfira, T., 2010. Expression, purification and characterization of cloning-grade zinc finger nuclease. J Biotechnol, Vol. 10, No. 1016
Towbin ,H., Staehelin, T., & Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.