Here is the entire set of questions: In this experiment you will be isolating pl
ID: 219808 • Letter: H
Question
Here is the entire set of questions:
In this experiment you will be isolating plasmid DNA by the alkaline lysis method and anion exchange, then submitting your purified sample for DNA sequencing by the Sanger method.
1) Describe the properties and the role of the following solutions used in plasmid DNA purification. Resuspension buffer, Lysis buffer, Neutralization buffer, Wash buffer, Elution buffer, Ion exchange spin-columns
2) What is the purpose of measuring the A260:A280 ratio for DNA samples? What is the acceptable range for your sample?
3) Imagine that your purified DNA has a concentration of 165 ng/uL. Describe how you would prepare this sample to send for DNA sequencing.
I'm asking for help on number three
I'm not quite sure where to start. With looking at the manual, it seems I have to start with a dilution- correct?
Table 1: Concentrations of the stock solutions of primers and prepared templates Template Type Size (kilo basepairs) [Templatel (ng/uL) [Primer] (pmol/uL) 0.100-0.300 0.301-1.000 >1000Explanation / Answer
Resuspension Buffer
The pellet is then re-suspended in a solution normally called solution I, containing Tris, EDTA, glucose and RNase A. Divalent cations (Mg2+, Ca2+) are essential for DNase activity and the integrity of the bacterial cell wall. EDTA chelates divalent 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.
The lysis buffer contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS). SDS is there to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and your plasmid, to single stranded DNA (ssDNA). 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.
Lysis Buffer
The lysis buffer contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS). SDS is there to solubilize the cell membrane. NaOH helps to break down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA (gDNA) and your plasmid, to single stranded DNA (ssDNA). 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.
Neutralization Buffer
Addition of potassium acetate decreases the alkalinity of the mixture. Under these conditions the hydrogen bonding between the bases of the single stranded DNA can be re-established, so the ssDNA can re-nature to dsDNA. This is the selective part. While it is easy for the the small circular plasmid DNA to re-nature it is impossible to properly anneal those huge gDNA stretches.
While the double-stranded plasmid can dissolve easily in solution, the single stranded genomic DNA, the SDS and the denatured cellular proteins stick together through hydrophobic interactions to form a white precipitate. The precipitate can easily be separated from the plasmid DNA solution by centrifugation.
5. Cleaning and concentration
Now 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, so it’s not much use for downstream applications. The next step is to clean up the solution and concentrate the plasmid DNA.
There are several ways to do this including phenol/chloroform extraction followed by ethanol precipitation and affinity chromotography-based methods using a support that preferentially binds to the plasmid DNA under certain conditions of salt or pH, but releases it under other conditions.
Rinse the resulting precipitate plasmid DNA in ice-cold 70% ethanol and let it dry for about 10 minutes to allow the alcohol to evaporate. You should likewise resuspend the DNA pellet in a buffer solution containing Tris, EDTA and RNases to cleave all the remaining RNAs in the solution.
The A260/A280 ratio is an indication of purity. Nucleic acids have absorbance maxima at 260. Historically, the ratio of absorbances at this wavelength has been used as a measure of purity in nucleic acid extractions. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. Similarly, absorbance at 230 nm is accepted as being the result of other contamination; therefore the ratio of A260/ A230 is frequently also calculated. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0–2.2.
To Send for Sequencing I will dilute the concentrated DNA with the sterile nuclease free water
to make a concentration of 60 ng/ul and then will send for sequencing
Yes, You will have to dulute the concentrated DNA with the sterile nuclease free water