Essay Example on The method of PCR was used for the rapid and specific amplification of DNA









The method of PCR was used for the rapid and specific amplification of DNA fragments of interest here pUC18 vectors with the fdx4 insert Besides restriction sites within the PCR primer sequences were built in for later cloning steps too The standard PCR protocol and the components for the PCR reaction which were used are shown in Table A1 Annealing temperature and elongation time were adjusted based on primer characteristics and the product size respectively PCR products were analyzed and separated by agarose gel electrophoresis These separated DNA fragments were excised and purified using a PCR purification kit 2 2 Agarose gel electrophoresis Agarose gel electrophoresis was performed to separate DNA fragments according to their size and therefore used to confirm each step of the cloning process Agarose gel was prepared by adding appropriate amount of agarose into TAE buffer samples were mixed with 6x loading dye including SYBR Green and the DNA fragments were separated in an electric field 80 120 V The agarose concentration of the gels was set to 1 0 according to the size of DNA fragments to be analyzed A 1 kbp DNA marker was applied as a size standard Upon finishing the gels were documented using a blue light transilluminator and a camera to take a photo If necessary DNA bands of interest were excised from the gel and the DNA was subsequently extracted using a QIAquick PCR Purification Kit according to the manufacturer s instructions 2 3 

Restriction ligation and transformation Purified PCR products were digested with the restriction endonucleases HindIII and NdeI and subsequently ligated using T4 DNA ligase into the corresponding expression vectors pT7 7 which were also digested with the same pair of restriction enzymes The ligation mixture was transformed into chemically competent E coli DH5 α cells by the heat shock method to amplify the plasmid DNA and plated on Lysogeny broth LB agar Each step was performed using suitable buffer systems which are listed with the other utilized components in Table A2 2 4 Colony PCR Additionally PCR was used to verify successful cloning Colony PCR was used as a fast method to identify and screen for positive transformed clones containing the vector with desired insert For a standard colony PCR reaction a single bacterial colony was picked up from a LB agar plate using a sterile toothpick and transferred into a PCR tube containing the PCR mix Table A1 PCR was performed immediately and the products were analyzed on an agarose gel with SYBR Green staining 2 5 Protein expression

Chemically competent cells of BL21 DE3 E coli strain were transformed with the expression vectors pT7 7 fdx4 extracted from DH5 α for protein production using standard methods An overnight 5 mL preculture received from a single colony from an agar plate was grown at 37 C and 220 rpm in LB medium supplemented with 100 µg mL ampicillin A 500 mL main culture was prepared as the starting culture including the required ampicillin and additionally a pinch of iron II sulfate was added and then inoculated by adding the preculture The cells were incubated at 37 C in the shaker When their OD600 reached 0 6 protein expression was induced by adding 0 1 mM isopropyl β D 1 thiogalactopyranoside IPTG The culture was left to grow at 37 C until cell culture density reached an OD600 of 5 Finally the cells were harvested by centrifugation 2 6 Cell lysis and protein purification Cell lysis was used as a method that disrupts cells and leads to the release of the containing proteins into the lysis buffer After cultivation the bacteria cell cultures were harvested by centrifugation 5 000 x g 4 C 15 min and the cell pellet was resuspended in lysis buffer

Table A3 with an amount of three times of the cell volume Cells were lysed twice with the EmulsiFlex cell disruptor and the lysate was centrifuged at 50 000 x g 4 C for 30 min to remove cell debris and high molecular weight DNA The resulting supernatant containing the soluble proteins was heated to 70 C for 10 minutes as this protein is from a hyperthermophilic organism and thus denaturing most of the E coli proteins which were removed by another centrifugation step 15 000 x g 20 C 10 min The supernatant was brought to 55 saturation in ammonium sulfate and the mixture was stirred at 0 C overnight The precipitated proteins were again centrifuged at 15 000 x g 20 C for 10 min and now the pellet was resuspended in original volume of DEAE loading buffer Table A3 for further purification by chromatography After each centrifugation cycle a small amount of the supernatant and the pellet was kept for further analysis by SDS PAGE 2 7 Chromatographic techniques All chromatography runs were performed using a Äkta Prime Plus GE Healthcare at room temperature For evaluation absorbance at 280 nm and conductivity were monitored and a chromatogram was recorded 2 7 1 Ion exchange chromatography IEX IEX separates proteins due to their affinity of reversible adsorption to a counter charged group immobilized on a matrix DEAE Sepharose After the column was appropriately equilibrated and A280 and the conductivity baseline stabilized AaFd4 diluted in lysis buffer was loaded onto the column Unbound material was washed with DEAE loading buffer Table A3 and subsequently the adsorpt protein sample was eluted with a flow rate of 2 mL min by gradually increasing the salt concentration with a linear gradient of DEAE elution buffer 0 mM to 500 mM NaCl Table A3 The eluates were collected in 2 mL fractions and analyzed by SDS PAGE

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