s (o points) a) in our plaamid punfication procedure, how did we lyse the petist
ID: 192419 • Letter: S
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s (o points) a) in our plaamid punfication procedure, how did we lyse the petist of bactial c b) What is another method we could have used to lyse our cells, and why do you k instead of these other possible methods (think about how the rest of our prolocr c) After lysing the cells, how did plasmid preps) proteins? be specific and explain the method ad we in our plasmid preps) manage to purity the plasmid DNA away trom cetllar ) Describe (name and briefly explain) one other methods we could have used to purity our plasmid ONA away om cellular proteins. ow did we separate our plasmid DNA from the chromosomal DNA and from the cellular RNA (two answers 1? inishing the procedure you were putting away the reagents and noticed that the buffer PE (the on wash the column) had not had the Ethanol added to it. Where do you think your plasmid DNA er ult of this mistake?Explanation / Answer
Plasmid are small circular genetic material in a cell which can replicate independently of a chromosome, It provides additional ability to a cell to survive in unfavorable condition (example resistance to antibiotics). In course of plasmid preparation from bacterial cell, bacteria is first lysed from either of two methods
Chemical method (Alkaline lysis method): it compromises three solutions named
Resuspension solution which is composed of Tris, EDTA, Glucose, RNaseA(optional can be used at the final step of plasmid purification),
Tris and EDTA chelates divalent metal ions such as magnesium and calcium, therefore, destabilizes the cell walls. It also inhibits the action of DNases
Glucose helps in maintaining the osmolarity of to keep the cell from bursting while RNAase A help in degrading the RNA once the cell is lysed.
The 2nd buffer is lysis buffer which comprises of NaOH and SDS, SDS is a detergent which causes cell wall to crack open whereas NaOH cause denaturation of gDNA and plasmid
The 3rd buffer is Neutralization buffer which consists of 5M potassium acetate, pH4.8, which helps readjust the pH and allow only the plasmid DNA to reanneal because the gDNA is so large, it cannot reanneal properly under these conditions
B) the second method of cell lysis is a physical method in which cell is resuspended in an appropriate buffer and subjected to high-frequency energy which agitates and disrupt the cells. However, use of ultrasonication may cause shearing of the plasmid as it produces lots of heat energy.
C) once the cell is lysed, it is centrifuged to remove the cell debris and we collect the supernatant. and subjected it to affinity chromatography. DNA has an affinity to bind to silica so we transfer collected supernatant into a spin column which has a silica bed.DNA bind plasmid binds with silica whereas rest of the unwanted molecules get pass through the bed. flow through is discarded and then plasmid present on the spin column is subject to washing using 70% ethanol solution which removes mostly salt bound with Plasmid. Column is air dried and then plasmid is eluted by incubating column using appropriate amount of either elution buffer or DNase free water
D) alcohol precipitation method can also b used for isolating plasmid after cell lysis. the cell lysate is the first subject to remove the cell debris that is then subject to centrifuge followed by phenol: chloroform: isoamyl alcohol (25:24:1 v/v) treatment, result in two-phase separation. the upper phase is the aqueous phase which contains the Plasmid. we carefully remove upper phase and collect in a fresh tube then we add an equal volume of ice-cold 100% isopropanol or 100%ethanol to precipitate the plasmid. we centrifuged it and pallet down the plasmid followed by washing with 70% ethanol followed by air dry and then pallet is dissolved in appropriate buffer.
E) NaOH in lysis buffer causes denaturation of both chromosomal DNA and plasmid. when we use Neutralization buffer which consists of 5M potassium acetate, pH4.8, results in a readjustment of the pH and allow only the plasmid DNA to reanneal because the gDNA is so large, it cannot reanneal properly under this conditions and hence precipitated down which then centrifuged to separate the chromosomal DNA from the plasmid.
RNase A is the enzyme which degrads RNA therefore seperate RNA from Plasmid.
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