Needing help understanding certain steps of the following PCR reaction experimen
ID: 164828 • Letter: N
Question
Needing help understanding certain steps of the following PCR reaction experiment:
1) Spin the tubes containing the O/N culture at the highest speed for 1 min. Note that for all spins the hinges connecting the caps to the tubes should be oriented outwards.
2) Remove enough supernatant to leave 0.4 mL in each tube; cap the tube and resuspend the cells using the vortex mixer. The removed supernatant should be added to a biohazard waste beaker.
3) Add 5 µL of Tide to each tube and vortex briefly to mix (about three, 2-second pulses).
4) Incubate the tubes at 37 °C for 10 min.
5) Add 750 µL of water-saturated n-butanol to each tube; vortex briefly to mix.
6) Spin at top speed for 5 min.
7) Carefully lower a clean 10 – 200 µL pipette tip through the organic layer and into the lower aqueous layer. You likely will have to push aside precipitated protein at the interface of the two layers. Slowly withdraw 200 µL of the aqueous layer, taking care not to disturb the pellet at the bottom of the tube or to siphon any of the precipitated protein at the interface, and transfer to a new microcentrifuge tube.
8) Add 700 µL of 2-propanol to each of the 200-µL aqueous samples. Gently invert 5 times to mix.
9) Spin at top speed for 5 min.
10) Carefully decant the supernatant into the waste beaker and drain tubes on a Kim-wipe. You likely will see a slightly yellow pellet at this point. You should know where to expect the pellet because of the manner in which you oriented your tubes in the microcentrifuge – see #1 above.
11) Add 700 µL of 70% EtOH to your tubes containing the pellets. Gently invert to mix.
12) Spin at top speed for 2 min.
13) Carefully remove supernatant with a drawn-out Pasteur pipette. Be very careful not to disturb the pellet, but make sure to remove all visible traces of liquid from the sides of the tube.
14) Dry the open tubes in the 37°C incubator for 5 min. or until you do not see any traces of liquid (*a critical step!).
15) To each tube, add 50 µL of a solution containing 0.1 mg/mL RNase A in autoclaved dH2O.
16) Incubate at 60 °C for 30 min. Midway through the incubation, remove tubes and gently tap the sides to dislodge any tightly adhering pellets.
17) Use 2µl sample to measure the absorbance at 260nm and 280nm using the “Take3” low volume accessory with the Lab Plate Reade
QUESTIONS:
a) What role does the Tide Free 2X Ultra play in the lysis of the cells? The detergent and lipases help break down cell membranes, the proteases help degrade cellular proteins including deoxyribonucleases?
b) What components of the cells are dissolved in the n-butanol layer?
c) Why is the vortex mixing at step 5 of the protocol so brief?
d) What step(s) likely helped prevent the degradation of the genomic DNA by deoxyribonucleases (DNases)?
e) What components of the cells formed the film at the interface between the organic and aqueous layers?
f) What causes the DNA to precipitate when 2-propanol is added at step 8 of the protocol?
I greatly appreciate it
Explanation / Answer
A) The detergent ( Tide Free 2X) in this case is indeed used to break open the cell and nuclear membranes and to remove the cellular contents in the aqueous medium. Addition of proteases is necesaary in order to inhibit the activity of nuclease ( DNAse, RNAse, Endo-nuclease etc.), so that the genetic material is not damaged.
B) The butanol is added, not to dissolve any component, but to precipitate the DNA ( which is insoluble). Any residual organic matter is dissolved in butanol.
C) Excessive vortexing can result in the formation of an emulsion, from which it would be difficult to separate DNA.
D) Treatement with detergent, repeated washing with ethanol and addition of proteases ensures that DNA is not damaged.
E) The degraded proteins and carbohydrates ( organic molecules) are found at the interface of organic and aqueous layers.
F) 2-propanol has a low dielectric constant. It accentuated the interaction between Na ions ( which comes from salt addition) and the phosphate group of DNA. This makes the DNA less hydrophlic and causes it to precipitate out of the solution.
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