QBM exam 3| Questions Solved with Verified
Correct Answers | Latest Update 2024-2025
DNA Quantification: - ANSWER - Next step after miniprep
- Spectrophotometer - 10X, 100X dilution, read absorbance at 260
- Nanodrop - 1 μl (usually 2 μl): use this one
- A260 of 1.0 = 50 μg/ml DNA
- A260/A280 > 1.5
- Won't work if there's too much DNA b/c enzymes won't work & it'll be too sticky
RNA Purification: - ANSWER - The isolation of intact, high quality, total cellular RNA is often
the starting point for many molecular biological procedures
- Acquire purified cellular RNA
- in vitro translation, cDNA library construction, Northern blot analysis, PCR analysis
RNA Purification Challenges: - ANSWER - More difficult to work w/ than DNA
- RNA is more susceptible to degradation than DNA, due to the 2' hydroxyl group
(nucleophile) adjacent to the phosphodiester linkages in RNA
- mRNA - capping, polyA tail hold off degradation for only a short time
,- DNases require metal ions for activity and can be inactivated with chelating agents (EDTA), but
- RNases do not use metal ions
- Really suseptible to cleavage
- Have to use special equipment
RNase: - ANSWER Single-strand specific endoribonuclease
- Found on your hands or in original tissue
- Resistant to metal chelating agents
- Can survive prolonged boiling or autoclaving
RNase activity is extremely difficult to inactivate
- Bake glassware at 280 °C overnight
- Treat water w/ diethyl pyrocarbonate (DEPC)
- Use molecular biology grade chemicals
- Wear gloves during all procedures
RNA Isolation: - ANSWER - Cell type and target RNA is relevant
- mRNA will comprise 2-5% of total RNA
- polyA tail binds to oligo-T beads or columns
- Different cell types can contain different RNA amounts
RNA Isolation Method: - ANSWER 1. Solubilize cellular components
2. Denature proteins
3. Remove lipids
,4. Inactivate RNases
Isolation procedures use one or more reagents to improve yield and quality of mRNA: -
ANSWER - Chaotropic salts, such as guanidinium
- isothiocyanate (GITC) to inactivate Rnases and organic solvents (phenol,
chloroform) (Chomczynski and Sacchi, 1987)
- Cesium chloride gradients
- Detergents (i.e. sodium dodecyl sulfate (SDS), N-lauryl sarcosyl, Nonidet P40,
sodium deoxycholate, Catrimox-14
Chomczynski and Sacchi: RNA Isolation - ANSWER - 1st to perform RNA isolation
- Uses guanidinium isothiocyanate to denature proteins and inactivate intracellular RNases
1. Harvest Cells
2. Add guanidinium thiocyanate
3. Add phenol + CHCl3
4. Centrifuge
5. Aqueous Phase
6. Remove aqueous phase
7. RNA, DNA + DNA protein
8. Add isopropanol
9. Centrifuge (RNA pellet)
10. Resuspend RNA in steile H2O
11. Total RNA
, Gel Electrophoresis of DNA: - ANSWER - Frequently used to monitor progress of many
DNA-based applications (PCR, digestion, mini preps)
- At alkaline pH, DNA and RNA strands are NEGATIVELY- CHARGED and therefore migrate in
an electric field
- For small DNA fragments, polyacrylamide gel electrophoresis (PAGE) can be used, and the
technique used is similar to the protein gel electrophoresis method w/o a stacking gel
- For larger DNA molecules (>200 bp), electrophoresis in agarose gel electrophoresis is
the method of choice
- Separates based on size ONLY
Agarose Gels: - ANSWER - TAE (Tris/Acetate/EDTA), TBE (Tris/Borate/EDTA), or Boric Acid
- Ethidium bromide (used in QBM lab) or alternatives
- Agarose - ~1 gram per 100 ml buffer (1% will be faster than 2%)
- Microwave
(faster than hot plate)
- Comb
- Power supply
- UV lamp
Correct Answers | Latest Update 2024-2025
DNA Quantification: - ANSWER - Next step after miniprep
- Spectrophotometer - 10X, 100X dilution, read absorbance at 260
- Nanodrop - 1 μl (usually 2 μl): use this one
- A260 of 1.0 = 50 μg/ml DNA
- A260/A280 > 1.5
- Won't work if there's too much DNA b/c enzymes won't work & it'll be too sticky
RNA Purification: - ANSWER - The isolation of intact, high quality, total cellular RNA is often
the starting point for many molecular biological procedures
- Acquire purified cellular RNA
- in vitro translation, cDNA library construction, Northern blot analysis, PCR analysis
RNA Purification Challenges: - ANSWER - More difficult to work w/ than DNA
- RNA is more susceptible to degradation than DNA, due to the 2' hydroxyl group
(nucleophile) adjacent to the phosphodiester linkages in RNA
- mRNA - capping, polyA tail hold off degradation for only a short time
,- DNases require metal ions for activity and can be inactivated with chelating agents (EDTA), but
- RNases do not use metal ions
- Really suseptible to cleavage
- Have to use special equipment
RNase: - ANSWER Single-strand specific endoribonuclease
- Found on your hands or in original tissue
- Resistant to metal chelating agents
- Can survive prolonged boiling or autoclaving
RNase activity is extremely difficult to inactivate
- Bake glassware at 280 °C overnight
- Treat water w/ diethyl pyrocarbonate (DEPC)
- Use molecular biology grade chemicals
- Wear gloves during all procedures
RNA Isolation: - ANSWER - Cell type and target RNA is relevant
- mRNA will comprise 2-5% of total RNA
- polyA tail binds to oligo-T beads or columns
- Different cell types can contain different RNA amounts
RNA Isolation Method: - ANSWER 1. Solubilize cellular components
2. Denature proteins
3. Remove lipids
,4. Inactivate RNases
Isolation procedures use one or more reagents to improve yield and quality of mRNA: -
ANSWER - Chaotropic salts, such as guanidinium
- isothiocyanate (GITC) to inactivate Rnases and organic solvents (phenol,
chloroform) (Chomczynski and Sacchi, 1987)
- Cesium chloride gradients
- Detergents (i.e. sodium dodecyl sulfate (SDS), N-lauryl sarcosyl, Nonidet P40,
sodium deoxycholate, Catrimox-14
Chomczynski and Sacchi: RNA Isolation - ANSWER - 1st to perform RNA isolation
- Uses guanidinium isothiocyanate to denature proteins and inactivate intracellular RNases
1. Harvest Cells
2. Add guanidinium thiocyanate
3. Add phenol + CHCl3
4. Centrifuge
5. Aqueous Phase
6. Remove aqueous phase
7. RNA, DNA + DNA protein
8. Add isopropanol
9. Centrifuge (RNA pellet)
10. Resuspend RNA in steile H2O
11. Total RNA
, Gel Electrophoresis of DNA: - ANSWER - Frequently used to monitor progress of many
DNA-based applications (PCR, digestion, mini preps)
- At alkaline pH, DNA and RNA strands are NEGATIVELY- CHARGED and therefore migrate in
an electric field
- For small DNA fragments, polyacrylamide gel electrophoresis (PAGE) can be used, and the
technique used is similar to the protein gel electrophoresis method w/o a stacking gel
- For larger DNA molecules (>200 bp), electrophoresis in agarose gel electrophoresis is
the method of choice
- Separates based on size ONLY
Agarose Gels: - ANSWER - TAE (Tris/Acetate/EDTA), TBE (Tris/Borate/EDTA), or Boric Acid
- Ethidium bromide (used in QBM lab) or alternatives
- Agarose - ~1 gram per 100 ml buffer (1% will be faster than 2%)
- Microwave
(faster than hot plate)
- Comb
- Power supply
- UV lamp
