MOLBIO - MT122 - Laboratory 6 - Nucleic Acid Extraction Part 2

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Class II BSC

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What biosafety cabinet is used for Nucleic Acid Extraction?

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b. Speed should be capable of 20,000 x g

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Which of the following is not true about Centrifugation?

a. Must have a removable rotor placed inside the BSC
b. Speed should be capable of 20,000 x g
c. Should have a sealing biocontainment lid or cover inside
d. It should push materials through the filter
e. None of the choices

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27 Terms

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Class II BSC

What biosafety cabinet is used for Nucleic Acid Extraction?

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b. Speed should be capable of 20,000 x g

Which of the following is not true about Centrifugation?

a. Must have a removable rotor placed inside the BSC
b. Speed should be capable of 20,000 x g
c. Should have a sealing biocontainment lid or cover inside
d. It should push materials through the filter
e. None of the choices

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Vortex

- It is used inside the BSC
- Thoroughly mix samples and reagents
- Variable speed

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Mini-Centrifuge

- Quickly spin MCTS (remove droplets from the lid)
- Used inside BSC

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Chaotropes

Safety Information:

Buffer NVL contains _________

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True

True or False: Sample containers should only be opened inside the BSC to avoid contamination

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- Buffer
- Carrier RNA

When opening a new kit for RNA extraction, these reagents should be prepared:
1. __________
2. __________

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True

True or False:

Purpose of Carrier RNA

- Carrier RNA is added to improve the binding capacity of the mini-spin column when viral nucleic acids included in the sample are low copy and also protects the target nucleic acids from the chance of degradation due to RNase or residual RNase activity

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- Left
- Right
- Left
- Center

Setting up the BSC

- Set up the work surface with clean tubes and reagents on the _____ side
- Set the waste container and dirty items on the_______ side
- Place vortex and mini-centrifuge on the ______ side of the cabinet
- Samples are placed in the _______

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300 uL

RNA Extraction Procedure

Sample Lysis

Add _______ of buffer NVL to each MCT

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7 uL

RNA Extraction Procedure

Sample Lysis

Add ____ of carrier RNA solution to each MCT alreadt containing buffer NVL

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350 uL

RNA Extraction Procedure

Sample Lysis

Add _______ of buffer RB1 to each MCT, mic by pipetting up and down 10x

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750 uL

RNA Extraction Procedure

Sample Lysis

Transfer up to ________ of the sample-buffer solution to a labeled mini-spin column,take care not to wet the rim of the spin column when transferring

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10,000, 30 seconds

RNA Extraction Procedure

Sample Lysis

Centrifuge at _________ x g for ____ seconds, loosen the rotor, and place back into the BSC

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500 uL

RNA Extraction Procedure

Washing

Add ______ of buffer RBW to the mini-spin column, take care not to wet the rim of the column

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1 minute

RNA Extraction Procedure

Washing

Place the spin columns/collection tubes back into rotor and centrifuge at full speed for _______

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500 uL

RNA Extraction Procedure

Washing

Add _______ of buffer RNW to the mini spin column, take care not to wet the rim of the column

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30 uL

RNA Extraction Procedure

Elution

Carefully add ______ of nuclease-free water to the
center of the mini-spin filter membrane, use caution not to touch the membrane with the pipette tip

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1.5 mL

RNA Extraction Procedure

Elution

Remove the mini-spin column and discard, retain the _______ MCT with extracted RNA

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-70°C

RNA Extraction Procedure

Elution

Proceed directly to PCR. Extracted RNA is extremely sensitive to degradation. Short term storage at 0°C and long term at _______

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False

-20°C

True or False: After reconstitution of carrier RNA with nuclease-free water, the carrier RNA solution should be stored at 2-8°C in aliquots for conservation of activity or used immediately for experiments.

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e. None of the above

Which of the following is not true about low yield?

a. Poor quality of starting materials
b. Inefficient or insufficient lysis
c. Improper elution
d. Incorrect use of carrier RNA solution
e. None of the above

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I, III, IV

Which of the following pairs is/are correct?

I. Poor quality of starting materials- Too old or improperly
stored sample often yield degraded DNA. Use fresh sample, if possible. Repeated freezing and thawing the sample should be avoided.
II. Precipitation of buffer NVL- Add carrier RNA solution at lysis step. Omission of Carrier RNA may lead to low purification efficiency.
III. Degradation of Carrier RNA- Carrier RNA should be stored at -20°C in aliquots after reconstitution
IV. Improper elution- Add nuclease-free water to
the center of the spin column membrane and perform incubation for 1 min before centrifugation

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True

True or False

DNA Extract Protocol

Visually inspect for a pellet at the bottom of the tube, at the side of the hinge. A pellet is not always visible, so you can choose to centrifuge again or proceed as normal.

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False

(250 uL)

True or False

DNA Extract Protocol

Clean the DNA pellet by adding 200 uL of 70% ethanol to each sample. Do not mix the solution as it is imperative that the pellet is not disturbed. Repeat the process for all remaining samples.

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True

True or False

DNA Extract Protocol

It is important to remove as much supernatant as possible without disturbing the pellet

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20- 30 minutes

Allow the DNA to air dry for _________minutes