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Sample of an unknown cultured bacterium is used to extract the genomic DNA by first cell lyses and then purification of the DNA (nucleic acid purification). The resulted DNA is then run on agrose by gel electrophoresis in order to check its purity. A band of low intensity was observed probably due to presence of contaminants. This DNA is then used as the template for PCR amplification. Once reaction is complete, it is purification is performed to remove the PCR reagents. The integrity of DNA fragment after PCR is again checked by electrophoresis which dint show a reliable result. These fragments of DNA are then incorporated into a vector to produce recombinant DNA molecule, following its insertion into a suitable host for further sequencing. The resulting recombinants were selected on basis of blue/white screening technique; however we were not able to obtain any colony.
Many different methods and technologies are available for the isolation of genomic DNA and its sequencing. The choice of a method depends on many factors: the requisite quantity, weight of the DNA, the purity required for downstream applications, expenses and the time.
The initial step of genomic DNA extraction is the most crucial step of the whole experiment, any error in this procedure will lead to either contamination or loss of desired DNA itself, thus unwanted results. Bacterial cell consists of an outer cell membrane, in order to release the
DNA from the cell; we need to add certain reagents that cause the breakage of cell membrane and lyse the cell. It also removes the protein and other unwanted cell debris, the DNA left is then purified further which involves the separation of RNA from DNA and removal of coagulated proteins(by proteases). Once pure DNA is obtained, a gel electrophoresis is performed in order to check the integrity of DNA sample, it separates the nucleic acid on the basis of size, charge and other physical properties, the results of electrophoresis show how much amount of total DNA is obtained after extraction. If the extraction worked, the DNA should be visible and should travel more slowly than any of the marker.
This sample of DNA is then amplified using a Polymerase Chain Reaction (PCR) which causes the amplification of desired fragment into large numbers. PCR uses a primer sequence to attach with the desired fragment of DNA to be amplified and causes it to multiply into many copies. The PCR product is then purified to remove the reagents, and visualized on a gel electrophoresis to check the amount of sample amplified during the reaction. A thick band is observed somewhere in the middle of the gel which shows the size of the fragment and that it has been amplified.
Sequencing procedure includes the incorporation of the desired fragment into a suitable vector (usually plasmids/phage vector). Restriction enzymes are used to cut the desired DNA and the vector DNA, these are then ligated together to form a single DNA. This recombinant DNA is then transformed into a bacterial cell for replication. An efficient cloning reaction produces several hundred colonies of the vector along with the DNA of interest. Final step involves the identification of the recombinant cell on Xgal using blue and white selective screening. The recombinants appear as white whereas the nonrecombinants are blue on Xgal agar plate. Presence of either white or light blue colony indicates that the gene of interest is been correctly incorporated into the vector. (B-galactosidase was not produced)
Material and method:
Genomic DNA extraction:
To release DNA from bacterial cell, in a microfuge tube add 200ul resuspension solution, 20ul RNase A (20mg/ml), 25ul lyzozyme (50mg/ml), 20ul Proteinase K (20mg/ml) and a loopfull of cells from agar plate, vortex to mix and incubate at 55C for 20min. Add 200ul lysis solution, vortex to mix and incubate at 70C or 10min.
Now add 500ul of column preparation solution to the column, microfuge at a max speed for 1min and discard the flow through.
For binding of DNA to column, add 200ul ethanol to cell suspension, vortex to mix, transfer the cell suspension to primed column, microfuge at max speed for 2min and discard the flow through. To remove contaminants add 500ul wash solution to column, microfuge at 9000rpm for 1min, discard flow through, add 500ul wash solution to column, microfuge again at 12000rpm for 3min, discard the flow through, now microfuge at max speed for 1min to dry the column.For elution of pure DNA transfer the column to new microfuge tube, add 200ul elution solution, microfuge at 9000rpm for 1min.
Electrophoresis: Preparation of running buffer 10X TBE: Tris 108g, boric acid 55g, Na4EDTA 9.34g (40ml of 0.5M EDTA) add H2O to give a final volume of 1litre. This was however performed by only few students.
Preparation of agrose gel:
Agrose 1g, 0.5X TBE 100ml, heat till fully dissolved. Cool to approximately 50C before adding 5ul of ethidium bromide solution (10mg/ml). Gently mix then pour into gel casting tray and allow solidifying.
Check the DNA integrity by electrophoresis and visualization of DNA on agrose gel.
PCR amplification of DNA:
Setup PCR reaction mix of 25ul
Use 12.5ul of 10X NH4 buffer, 2ul of dNTP mix (12.5mM), 2.5ul of forward primer 27f (20uM), 2.5ul of reverse primer 1525r (20uM), 7.5ul of MgCl2 (50mM), 95ul of sterile water, 1.25ul of taq polymerase and 1ul of genomic DNA, mix well by vortexing and microfuge briefly.brady lane.JPG
Set condition of PCR and run: 95c for 45sec, 46c for 30sec, 74c for 40sec and total of 35 cycles.
Purification of PCR products: To 5ul of PCR products add 2ul of exoSAP-IT, mix and incubate at 37c for 15min, inactivate exoSAP-IT by heating at 80c for 15min.
Sequencing ââ‚¬" cloning PCR product:
For ligation of DNA add 2ul of PCR product to 1ul of salt solution, 2ul of sterile water and 1ul of TOPO vector.mix and incubate at room temperature for 10min.
For transformation add 2ul of cloning reaction mix into Ecoli and mix gently, incubate on ice for 20min, then heat shock the cells for 30sec at 42c, immediately transfer the cells into ice and add 250ul of LB medium at room temp.
Now shake the tube at 200rpm for one hour at 37c. Spread 100ul onto a selective plate and incubate over night at 37c. Check the plates for blue and white colonies the next morning.
DNA extraction and gel electrophoresis to check its purity: (the diagram of the gel is shown below)
Figure : Lane 2 shows our DNA on the gel.
DNA was obtained and is shown (lane 2). Due to the presence of another fainter band at the bottom of the lane the integrity of the DNA is proven to be less. In addition to this the band itself is lighter which is because the DNA has a low concentration. The lighter band (lane 2) shows that the DNA was contaminated prior to loading, if we compare our band with the band of 8th lane, we can clearly see the difference in concentration of dark band. The presence of the band at the bottom usually refers to the presence of RNA in the sample as a major contaminant; this is because RNA travels the furthest in the electrophoresis gel. Since we did not achieve a DNA it is not possible to measure the size; therefore we have obtained the size from fermentas website: (The bands indicate the distance (cm) travelled by the DNA sequences according to their fragment sizes (bp)).
Figure : Picture showing the sizes of DNA and there distances on the gel, SOURCE: www.fermentas.com
Gel electrophoresis to visualize the PCR amplification results:
Figure : Gel electrophoresis of DNA after PCR, our lanes are: 6,7,8,9
PCR performed to amplify the sequence of interest. Our PCR results are in the lane 6, 7, 8 and 9. The PCR resulted showing no significant difference between any of the four lanes and there is no band present in the middle region, which shows that there is no amplified fragment present. There are various reasons why PCR might not work that include incorrect pipetting and/or presence of an inhibitor during DNA preparation. However the only band visible is the lower band which proves the presence of the contaminants. Comparing our sample lanes, to those of 10, they have DNA in high concentration.
Cloning and sequencing of PCR product:
During the PCR amplification step, there was either no amplification of DNA fragment, thus when we performed the cloning step and plated on the agar with X-gal, we were not able to obtain any colonies. There were no light blue or dark blue colonies from our group. This clearly shows that there was no DNA present and the procedure followed for incorporation and transformation of colonies was not correct. If the experiment worked out properly one colony would be enough to interpret the results.PCR Gel B.jpg
Even thought we did not obtain any results, we were sent the DNA sequences (FASTA file). Following Neighbors-joining tree shows that the organism pseudomonas fluorescens, Compared to the other related organisms and strands from the Blast search, pseudomonas fluorescens is of the highest evolutionary class. (See figure 4)
The procedure for the DNA extraction and its purification is highly crucial since all the remaining results are dependent on it. During the extraction of DNA from the bacterial cell our DNA got contaminated either due to the presence of RNA or other protein contamination. When gel electrophoresis was performed to check the purity of the DNA, a light band was observed (as shown in the diagram above) this proved that we initially did not have much DNA in our sample (we can compare our band with another band having a dark intensity), in addition to this the presence of a lower band showed RNA contamination. If the experiment was carried out carefully and extraction was successful, we would have seen a single dark band on the top of the gel which moves slower than the marker, with no other bands, indicating the presence of DNA in high concentration.
Figure : Phylogenetic tree showing the root for P. fluorescens, SOURCE: NCBI - BLAST Using the same sample as a template for the PCR amplification procedure, we saw that there were no fragments observed after amplification, this is because the PCR was not able to amplify our DNA since the initial concentration was less, due to contamination. This was seen when the sample was visualized after gel electrophoresis of purified PCR product. If the initial step resulted in pure DNA at a high concentration, PCR reaction would have amplified the DNA fragment and thus a thick band would have had appeared somewhere in the middle of the gel (a 1500bp product), however in our gel we did not observe any band.
The Final step involved the cloning of desired amplified DNA fragment by incorporation into a suitable vector and integration of this recombinant DNA into a host cell for division. In our experiment we just got no clear colony, there were no light blue or dark colony from my plate, or any one in my group. This is again obvious since there was no DNA for successful incorporation into the vector. If the experiment is conducted properly and recombination occurs, we observe the presence of either white or light blue colony that indicates that the gene of interest is been correctly incorporated into the vector, replacing the b-galactosidase gene (LacZ gene) with itself, as this happens no b-galactosidase enzyme is produced on Xgal and thus it forms white colonies. Similarly if the gene of interest is not incorporated on the LacZ gene region, it causes the production of b-galactosidase enzyme which in turn causes the appearance of colonies as dark blue. But since the overall procedure was followed to be incorrect we were not able to see any colony on the plate. Therefore re-conducting the whole experiment would be the only way to identify the unknown organism and its position in the phylogenetic tree.