Term
| What is the purpose of a detergent in DNA isolation? |
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Definition
| To attach an emulsify lipids |
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Term
| During organic dna extraction, how can one remove cellular debris/proteins/lipids/carbs? |
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Definition
| High salt, low pH, mixtures of phenol and chloroform |
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Term
| During organic dna extraction, how is DNA ultimately collected? |
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Definition
| It is precipitated with ethanol or isopropanol in salts. Then centrifuged and rinsed |
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Term
| What is a common name for inorganic dna extraction? |
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Definition
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Term
| How does inorganic dna extraction work? |
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Definition
| uses the same low pH and high salt concentration as seen in organic extraction. However, the DNA is left in the solution, precipitated with isopropanol, and then resuspended in a buffer or water. |
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Term
| What is the benefit of inorganic extraction over organic extraction of dna? |
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Definition
| You avoid the biohazard of exposure to phenol |
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Term
| With RNA isolation organic extraction, how does lysis occur? |
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Definition
| By adding a detergent or phenol |
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Term
| Why is an RNAase inhibitor used during organic rna isolation? |
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Definition
| To prevent breakdown of the rna to be studied |
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Term
| During nucleic acid electrophoresis, what group ionizes, and why is this important? |
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Definition
| The phosphate group ionizes giving an overall negative charge which allows it to migrate towards the positive anode. |
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Term
| In what way does media affect particles during electrophoresis? |
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Definition
| It affects particles according to size so the electrical current effects the separation |
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Term
| What is the relationship between agarose and particle size during electrophoresis? |
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Definition
The more concentrated the agarose determines the size of the particels that can be separated.
More concentrated resolves smaller particels |
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Term
| What are the uses of polyacrylamide gel during electrophoresis? |
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Definition
| Used for smaller fragments, ssDNA, individual nucleic acids, mutation assays |
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Term
| What determines the quality of the polyacrylamide gel during electrophoresis? |
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Definition
| Determined by T and C (cross linking molecules) |
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Term
True or False:
Polyacrylamide gel provides good sensitivity to small amonunts of DNA |
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Definition
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Term
| How can one alter the size of the pores using polyacrylamide gel electrophoresis? |
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Definition
| By altering the cross linking molecules T and C. |
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Term
| With polyacrylamide gel electrophoresis, if you increase T, what happens to the pore size? |
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Definition
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Term
| Which fluorescent dye is known as an intercalating agent? |
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Definition
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Term
Which fluorescent dye does this describe:
When excited by light at 300 nm, emits light at 590 nm. |
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Definition
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Term
True or False:
Ethidium bromide is 25-100 times more sensitive than EtBr/ |
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Definition
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Term
|
Definition
Does not intercalate
sits in minor groove of double helix |
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Term
| When is it acceptable to use silver staining in regards to dna/rna extraction? |
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Definition
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Term
| Why laboratory stain is good for protein analysis? |
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Definition
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Term
| What two chemicals is a sample fixed with when using a silver staining technique? |
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Definition
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Term
| Once all of the chemicals have been added in a silver stain technique, how does the reaction continue? |
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Definition
| Silver ions react with acid resulting in the deposit of silver ions on the target molecules |
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Term
| Compare silver nitrate with silver diamine. |
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Definition
Silver nitrate - more stable
Silver diamine - best for thick gels |
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Term
| When quality checking DNA, what should the ratio be for a pure sample? Contaminated sample? |
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Definition
260/280 = greater than 1.6
260/280 = less than 1.6 indicated protein contamination (trp) |
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Term
| If an RNA quality ratio is 2.0-2.3, what does that say about the purity? |
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Definition
| The sample is very pure and good for use |
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Term
True or False:
SyBr green is a mutagenic substance |
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Definition
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Term
| What type of groups does a silver stain react with? |
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Definition
| Acidic or nucleophilic groups |
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Term
True or False:
Silver stain possess no biochemical harm |
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Definition
False
Silver stain is a biohazard |
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Term
| Describe the binding of SyBr green with RNA. |
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Definition
| Binds less well to RNA with poly A tial than with total RNA |
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