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pBluescript SK is a high copy plasmid. What does it mean? How would you purify low copy plasmid of a similar size?
A high copy plasmid pBluescript SK means that in the host organism, it is good at expressing recombinant genes and can self replicate better than low cop plasmid. With this you can get large plasmid numbers from relatively less cells in a shorter time frame.
Low copy plasmid of a similar size can be purified using a plasmid preparation. There are generally three steps involved; Bacterial Culture Growth, Bacteria Harvesting & Lysing, and Plasmid DNA Purification. Liquid cultures of bacteria which have undergone transformation and isolation have plasmids purified from them. A selectable marker is used which allows uninhibited multiplication of successfully transformed bacteria. Under alkaline conditions, precipitation of DNA and proteins occurs when bacteria are lysed. Many kits are available for purification, where for low copy plasmids, midi- and maxi- prep kits are commonly used. Proteins can be denatured and dissolved when phenol or chlorophorm are added.
Describe the major features of the pBluescript SK vector using the provided vector map (origin of replication, antibiotic resistance gene etc). Write about functional role of each of these structural features.
The pBluescript SK vector consists of a few structural features. The first one is the F1 phage intergenic region. There is also the LacZ region is the LacZ gene's 5' terminal part which encodes Î²-galactosidase's N-terminal fragment. Recombinant phagemids are able to have blue/white screening carried out on them due to this fragment. Phagemid replication is carried out by the rep (pMB1). At position 1213, replication of DNA is initiated, and then moves forward in the specified direction. Î’-lactamase is coded by the bla (ApR) gene, conferring ampicillin resistance. A signal peptide is coded for by nucleotides 2833-2765.
Two orientations of the Multiple Cloning Site within the LacZ region are represented by this pBluescript SK vector. The F1 (IG) region clones that are needed for the initiation for cis, DNA synthesis of phage F1 termination and DNA packaging into bacteriophage particles have their orientation indicated by the (+) and (-) symbols on this plasmid. Gene's II, V and X proteins that have been encoded by phage are essential for single stranded DNA synthesis, being initiated at ori (+), moving forward in the direction that is specified. Double stranded DNA being converted into single stranded DNA has no requirement of functioning phage genes. Beginning at ori (-), the RNA Polymerase of the host is synthesises a RNA primer of 30 nucleotides, causing initiation of DNA synthesis.
Which antibiotic would you use to grow E.Coli cells transformed with pBluescript SK plasmid.
Ampicillin is used to grow transformed E.Coli cells. Normally, in ampicillin presence, E.Coli would die, but transformed E.Coli are able to grow on ampicillin containing medium as the products of the ampicillin resistance gene are expressed from the plasmid by E.Coli. This is known as selection.
Explain what you observe at each stage of the plasmid DNA purification. What is the basis for this plasmid DNA purification technique?
Initially cells are centrifuged to obtain a pellet. Using a modified version of the alkaline lysis procedure, the cells were lysed. DNA was lysed and denatured after the cells pH was increased in this modified alkaline lysis method. The solution of DNA was then neutralised. Due to the circular and super coiled nature of plasmid DNA, DNA reverts back to its double stranded form when the pH is changed back to neutral.
No matter what happens, due to the largeness of genomic DNA, it's broken down in to linear pieces. In alkali, this is denatured, causing formation of precipitation in the lowered pH, whereupon being removed by centrifugation. Plasmid DNA is then purified additionally as to the silica gel membrane, supernatant is applied. DNA/RNA of a single strand fail to attach on to the membrane whilst under suitable conditions, double stranded DNA does attach. This causes organic solvents use elimination, allowing the removal of contaminants via the washing of DNA. Buffer low in ions is then used for elution of DNA. The plasmid DNA is now purified allowing for use in DNA sequencing.
What does "sense" and "antisense" orientation of thing1 gene relative to T7 promoter within pBluescript SK cloning vector means? Plasmid with what orientation of thing1 gene (sense or antisense) would you use to synthesize thing1 probe by in vitro transcription with T7 RNA polymerase for Northern and in situ hybridization analysis of thing1 gene expression?
The thing1 gene is present in the Multiple Cloning Site between the T7 and T3 promoters (626-791 BPs) relative to the pBluescript SK cloning vector. "Sense" relates to when the thing1 gene's start point is nearest to the T7 promoter. With regards to "antisense", this means when the end point of the thing1 gene is nearest to the T7 promoter.
A decision must be taken as to whether sense or antisense transcripts are required during the transcription template design. Complementary antisense transcripts are needed when RNA is going to be used as a hybridisation probe to mRNA in Northern Blotting or in situ hybridisation. When carrying out functional, expression or structural studies, or when a RNA quantitation standard curve is being formed with the use of an artificial sense strand RNA, sense strand transcripts are needed.
Why a restriction enzyme preparation is viscous. What is a "star" activity of a restriction enzyme and how to avoid it?
Restriction enzyme preparations are viscous because they're stored in 50% glycerol solution, which is viscous in itself. Also, genomic DNA from bacteria has been found to be viscous, however as bacterial DNA is degraded, there is a decrease in the overall viscosity. Viscosity could also be due to freezing and thawing, since these restriction enzymes are stored at -20°C, whilst the reason could be due to the difference in the extraneous nucleases, it's more likely that it could be due to the continual changes in the structure of the enzyme.
Similar sequences that are defined to their recognition sequence can be cleaved by some restriction enzymes under non-standard reaction conditions. This is known as "star" activity. This activity has been suggested to be a standard property of restriction endonucleases, and under some extreme conditions, they've been seen to cause noncanonical sites cleavage. The enzymes and conditions of the reactions inducing star activity are dependent upon in which the manner of enzyme activity alteration occurs.
There are a few ways of preventing star activity:
Don't use high concentration of glycerol. For the storage of restriction enzymes, 50% of glycerol is used; therefore the enzyme amount used should not be more than 10% of the reaction volume in total.
Avoid a high DNA ration enzyme/µg concentration. This can avoid and reduce over digestion and the reaction final concentration of glycerol respectively.
Use the optimal buffer to set up reactions as non optimal ones may contribute to star activity.
Try to keep reaction times to a minimum as long reaction times can cause star activity.
Organic solvents need to be kept out of/away from the reaction that may be present in the preparation of DNA.
Mg2+ should be used as the divalent cation. Restriction enzymes active site may not accept other divalent cations, causing interference with recognition.
Calculate restriction fragment sizes for all restriction digestions you did during the DNA workshop. What restriction reactions allowed to determine the orientation of thing1 gene insert? Explain your gel results. What other technique besides restriction analysis can you use to determine the orientation of thing1 gene in the plasmids if you know the complete DNA sequence of the 1.9 kb HindIII fragment containing thing1 gene.
Plasmid B restriction of EcoR1 produced two bands whereas plasmid A produced three bands when EcoR1 was cut meaning HindIII was cut before EcoR1 in plasmid A, however in plasmid B when EcoR1 was cut it produced two bands showing that it was cut before the HindIII was cut, suggesting that this is in the anti sense orientation, and plasmid A is sense orientation because EcoR1 is furthest away from the T7 promoter, whilst HindIII is closest to the same promoter.
Our results should have shown that the plasmid B EcoR1 produced the largest band (5000b), along with a second band at 700 bases. Plasmid A EcoR1 and both Plasmid A and B EcoR1 and HindIII containing lane should have produced three bands, with the largest at around 3000 bases. Both plasmid A and B that contained HindIII only should have shown the same results also with the largest band at 3000 bases and a second band at 2000 bases. The pBluescript plasmid should have shown a band at 3000 bases also.
Our actual results were similar, however there were some difference. The markers which were used were wrong, therefore these marker bands could not be related to with regards to this experiment. Also, the lane containing plasmid B EcoR1 differed from what was expected. It showed the same result as that for the lane containing both EcoR1 and HindIII for plasmid B. This could've been due to both plasmids being inserted into the ependorf tubes during the preparation of the samples.
A PCR primer binding within the vector which is near the site as to where the insert has been introduced can be used to determine the orientation of the thing1 gene if the complete DNA sequence of the 1.9 kb HindIII fragment containing thing1 gene is known. Towards one end of the already introduced insert, another PCR primer is required within that. In one of the orientations, A PCR product of known size will be produced, as the newly inserted primer will succeed with the vector's primer. However, a PCR product won't be formed is the insert is in the opposite direction, as the primer in the vector won't be bound to by the PCR primer, since the PCR primer will be pointing away from the vector's primer. Two PCR's can also be set up, so that in one orientation of the insert, the first PCR will work, whilst also the second PCR will also work if the insert is in the opposite orientation.
Explain the principles of DNA fragment separation in the agarose gel. What the major component of a loading buffer? How the DNA fragments are visualized.
After extraction of genomic DNA from tissue, a restriction enzyme is used to digest it, causing various sizes of segments to be formed via cleavage. Agarose gel electrophoresis can be used to separate these segments. These segments are then detected by a molecular probe which has similar sequences. A high voltage is used to move these fragments in agarose gel electrophoresis. The fragments movement rate is inversely correlated with fragments size; therefore you'd expect to see the heavier and lighter fragments move to and away from the site of loading respectively.
The major component of a loading buffer is to monitor the progression of the gel run via the density and dyes as it contains ficol or glycerol. Ethidium Bromide can be used for the visualisation of DNA fragments as well as other intercollating dyes. DNA fragments fluoresce when Ethidium Bromide is intercollated with them after being exposed to UV light.
The thing1 gene was cloned as a HindIII fragment into HindIII site of pBluescript SK plasmid. What would you do to reduce number of clones containing self ligated pBluescript SK plasmids ("self ligation" background) during this process? Explain how you can use colour selection to pick up clones containing your target plasmids and not "empty" pBluescript SK vector during this cloning.
In order to reduce clones containing self ligated pBluescripe SK plasmids, before ligation, dephosphorylation of the vector will help. Also, with the use of two restriction enzymes producing two different ends ensuring that there is only one desirable orientation of the insert can also solve this problem of self-ligation.
Colour selection can be used to pick up clones containing the target plasmids and not empty pBluescipt SK vectors during this cloning as this method is for identifying bacterial clones which contain plasmids with inserts. The pBluescript SK vector has its polycloning site as part of the galactosidase encoded by the LacZ gene, providing complementation. Blue colonies will be produced when re-ligated (empty) vectors are grown on IPTG and X gal containing plates. However, white colonies are produced when a functional galactosidase isn't able to be produced by colonies containing a substantial within the polycloning site of the plasmid.
Additionally, in this practical, the purity of the DNA sample was calculated. The maximum absorbance of DNA is 260nm. DNA purity is reflected upon by the ratio when the absorbance at 260nm id divided by the absorbance of 280nm. A ratio between 1.7-2.0 is good for a DNA sample.
DNA purity was calculated as follow:
A260= 1.016 AU A280= 0.788 AU
DNA Purity = A260 ÷ A280 = 1.016 ÷ 0.788 = 1.2894
Due to the DNA purity not being within the "good" range, this shows there might have still been some contamination since the DNA is not considered to be put at 1.2894.
Purified DNA's concentration was also determined, as follows:
Plasmid DNA Concentration (µg/ml) = 71 x OD260nm units x 40µg/ml
Plasmid DNA Concentration (µg/ml) = 71 x 1.016 x 40 = 2885.44 µg/ml