3 RNase A. It is this solution

3 fragments

Intro A restriction enzyme is an enzyme produced only by
bacteria it has the ability to cut or cleave DNA at specific sites / nucleotide
sequences of the DNA. Bacteria produce these enzymes in order to remove virus
particle’s viruses from DNA. Bacteria has what is called plasmid DNA which is a
circular double stranded piece of dna which can replicate independently, this
is in addition to its chromosomal dna. Plasmids usually offer functional
benefits to hosts such as antibiotic resistance or virulence amongst other
benefits. Within the plasmid DNA there is an origin of replication which allows
initiation of replication as well as many other different sites that allow for
different types of lab applications to be carried out. Restriction enzymes are
used in lab experiments for analysis via DNA fragmentation and are now a basic
tool in most biotechnology labs and research. Pbr322 cloning vector can be
inserted into microbes, in order to autonomously replicate in a short space of
time to produce multiple copies for analysis. (cox et al 2012). Pbr322 has
selectable markers such as antibiotic resistance genes which can be used to
distinguish recombinant cells under external pressure where non-transferable
cells would otherwise die. Pbr322 also exhibits restriction sites. It is within
these restriction sites that DNA can be incorporated to form the recombinant
dna. Restriction sites are sites of the DNA strand that can be read by a
certain enzyme, made by the cell itself. The enzyme can read these sites as own
DNA when methylated, on the other hand DNA that is foreign is unmethylated and
read so, therefore can be distinguished and removed. This process evolved to
tackle the problem of foreign virus DNA. Resuspension After the cells are
harvested they are re suspended into a solution named P1, usually this solution
contains, Tris, EDTA, glucose and RNase A. It is this solution that compromises
the integrity of the cell walls. EDTA helps to stabilise cations in the
solution preventing DNases from damaging the plasmid and also helps by
destabilizing the cell wall. Glucose maintains the osmotic pressure so the
cells don’t burst and RNase A is included to degrade cellular RNA when the
cells are lysed. Lysis The lysis buffer sodium hydroxide which is the alkaline
and Sodium Dodecyl (lauryl) Sulphate (SDS). Which is used to dissolve the cell
membrane. Sodium hydroxide helps to disintegrate the cell wall as well as
disrupts the hydrogen bonding between the DNA bases. This converts the
double-stranded DNA in the cell to single stranded DNA. This process is called
denaturation and is central part of the procedure, which is why it’s called
alkaline lysis. SDS also denatures most of the proteins in the cells, which
helps with the separation of the proteins from the plasmid later in the
process. Centrifugation This method is used to separate the larger denser
material, in this case cell debris and chromosomal DNA from the supernatant
fraction. The centrifuge is spun so that the denser material travels in a
radial direction to the bottom of the test tube caused by the centripetal
acceleration. Neutralisation So at this stage in order for the genomic DNA to
re-nature we need to reduce the alkalinity of the mixture, lowering the ph. of
the solution and we would do this by adding a neutralisation buffer (potassium
acetate). With gentle mixing it is at this point that the genomic DNA will
precipitate and the plasmid DNA will stay in the solution. Vigorous mixing
should be avoided as it can shear the genomic DNA causing contamination and
this would be useless to us when it comes to the electrophoresis stage. The
supernatant contains the plasmid DNA and soluble proteins. Cleaning The plasmid
DNA has been separated from the majority of the cell debris but is in a
solution containing lots of salt, EDTA, RNase and residual cellular proteins
and debris. Repeated washing with a high salt buffer and a filter will remove
this debris and residue and can be discarded. You will be left with plasmid DNA
in the filter. This is the melting temperatures of the primer, which is usually
no more than 6oc – 8oc higher than the primers annealing temperature. If you
know the tm of each primer you can use a temp to ensure that both primers
anneal. If the tm difference of the two primers is too great it may be
difficult to manipulate lab conditions to ensure both primers anneal
successfully to the template. Primers with a melting temp of more than 50C or
greater than 70C usually are the least efficient. If the tm of each primer is
too alike self annealing may also occur causing hair pin loops in the amplified
DNA. If the difference is too high no pcr will take place. Self complimentary
Self complimentary is the score given to a primer which is the likelihood that
the primer will bind to itself. The scoring system gives 1.00 for a match,
-1.00 for a mismatch. The self-complementarity score is useful to predict
possible secondary structures between the primers itself or even primer pairs.
Ideally the lower the score the better. If the primer was to bind to itself,
amplification of would be unsuccessful. 3′ complimentary This is the score
given to a primer which is useful to predict the occurrence of primer dimers
and are a result of poorly designed primers. They occur when the 3′ of each
primer have complementary base pairs and when pcr takes place, instead of
amplifying the DNA the primers would just anneal. The results would be very few
bps of the primer dimer formation and no amplified DNA. Hetra-primer dimer
occurs when two primers are complimentary and anneal at the 3′ end. Homo-primer
dimer is a result of self annealing of head to tail. The lower the 3′
complimentary the better. Optimal range for primers The optimal range for
primers is between 18-30 bps. Any longer and it would increase the chance for
errors and long enough for suitable specificity. Longer primers require higher
annealing temperatures and would be less efficient during the annealing process
and therefore the pcr process would be have a higher chance of being less
successful. Any shorter and the bps may not be specific enough, and again would
increase the risk of an unsuccessful annealing process. GC% score/ GC clamp A
GC clamp is having either a G or a C base in the last 5 bases of the primer.
Since G and C bases have a stronger binding affinity, this will ensure the 3′
end of the primer anneals correctly to the cDNA sequence. GC bps are stronger
because they have three hydrogen bonds rather than two like the AT bonds. The
higher the GC% would indicate a more successful pcr process down to the
increased strength of the bonds

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