How do you prepare a solution in plasmid isolation?
Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15 minutes at 15 psi and store at 4°C. 1. 10 N NaOH stock solution (50 mL) Dissolve 20 gram of NaOH in 50 mL sterilized de-ion water.
What is the function of solution 1 in plasmid isolation?
Solution 1 contains glucose, Tris, and EDTA. Glucose is added to increase the osmotic pressure outside the cells. Tris is a buffering agent used to maintain a constant pH ( = 8.0). EDTA protects the DNA from degradative enzymes (called DNAses); EDTA binds divalent cations that are necessary for DNAse activity.
Which solution is used in plasmid DNA isolation?
This solution contains sodium hydroxide and SDS (sodium dodecyl sulfate). The sodium hydroxide denatures the plasmid and chromosomal DNA into single strands.
What are the different steps in the isolation of plasmid?
Procedure
- Harvest Bacterial and Resuspended Cells.
- Cell Lysis.
- Neutralization.
- Load Lysate on Column.
- Bind and Wash.
- Plasmid Elution.
How do you prepare solution 2 for plasmid isolation?
Procedure:
- Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria.
- Pour 1.5 ml of the culture into a microfuge tube.
- Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.
Which step of plasmid isolation contains a detergent?
The lysis buffer (aka solution 2) contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl (lauryl) Sulfate (SDS).
Why is sodium acetate used for plasmid isolation?
In some extractions such as plasmid preps, it is used to neutralize the alkaline component of the lysis (step 2 NaOH and SDS) and precipitate the proteins and genomic DNA from this step, again through ionic strength.
Why is NaOH used in plasmid extraction?
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).