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molecular-L midterm
molecular lab midterm prep
| Question | Answer |
|---|---|
| What is the purpose of suing organic solvents such as phenol-chloroform-isoamyl alcohol for DNA extraction? | To purify gDNA to obtain high quality gDNA. |
| What is called the separation method of phenol-chloroform isoamyl alcohol extraction? | liquid/liquid phase separation or biphasic purification |
| How many phases can you obtain by phenol-chloroform-isoamyl alcohol extraction? | 3 |
| what will you see and find in the top layer of P:C:I separation? | aqueous layer contains gDNA |
| What will you see and find in the mid layer of P:C:I separation? | Interphase contains proteins and phenol. |
| What will you see and find in the lowest layer of P:C:I separation? | Organic phase contains lipids and proteins and phenol and chloroform. |
| What is the function of phenol in P:C:I separation? | it is responsible for dissolving proteins. |
| What is the function of chloroform in P:C:I separation? | it is responsible for dissolving lipids and separating the aqueous and organic phase. |
| What is the function of Isoamyl alcohol in P:C:I separation? | it prevents fizzing, bubbling and forming of phenol and stabilizes the separation of aqueous and organic phases. |
| What is the difference between Triton X100 and SDS? | Triton X100 is milder detergent and has no charge. It can lyse RBCs but not WBCs. SDS is stronger, cationic detergent and can lyse more complicated cell membrane of WBCs. |
| How detergent lyse cell membrane? | itt dissolves membrane by denaturing(unfolding) proteins. |
| Why Proeinase K, which is also protein, cannot be destroyed by SDS? | SDS activates proteinase K by 20%. |
| What components "lysate" has? | It contains gDNA, lipids, proteins, RNA |
| Why P:C:I mixture is pH8 regardless of the acidity of phenol? | Because the mixture contains TE buffer(Tris EDTA). |
| Why P:C:I mixture needs to be pH8? | Because acidity breaks double strand DNA, especially gives effect on purines(Adenine&guanine) |
| What does RBC lysis buffer solution contain? | It contains Tris-HCL, sucrose, MgCl2, TritonX 100. |
| What role does sucrose in RBC lysis buffer take? | It creates hypertonic environment that will chrivel or crenate RBCs. |
| What does cell lysis solution contain? | It contains SDS, proteinase K buffer, Proteinase K enzyme. |
| Calculate the volume of methanol and gracial acetic acid when you make 40mL of 3:1 Carnoy' fixative with methanol and gracial acetic acid. | methanol: 30mL gracial acetic acid:10mL |
| Regardless of the source of gDNA, extraction procedures utilize the same key steps. List those 3 steps. | 1. lysis of the cell membrane, 2. Precipitation of gDNA, 3. Purification of the released gDNA |
| For lysis of the cell membrane, typuically ________ is used to break up the phospholipid bilayer. | a detergent |
| ___________ is added to digest released proteins. | Proteinase K |
| ________ is a chelator that binds up divalent cations. | EDTA |
| _________ serve as cofactors for nucleases tha degrade DNA | divalent cation such as Mg++ |
| In lysis solution, __________( to break up the membrane), __________(to digest proteins), and __________(to binds up divalent cations) are typically contained. | detergent, proteinase K, EDTA |
| what is the function of chelator? | bind up divalent cations |
| What are divalent cations? | they serve as cofactors for nucleases that degrade DNA |
| Alcohol allow DNA to _____________ out. | precipitate |
| NaCl ______A______DNA's ____B________ and allow DNA to aggregate and precipitate out of the alcohol solution. | A. neutralize B.net negative charge |
| The 4 major components of of blood are: __________, ___________, __________, __________. | RBCs, WBCs, platelets, plasma |
| Which component of blood makes up a significant portion of the total blood? | RBCs |
| Which component of blood has a nucleus? | WBCs |
| Which description on the Serological Pipette means "the pipette needs to be blown out for the volume"? | TC |
| What is the role of sodium acetate(NaOAc) in P:C:I extraction? | positive charge of Na in sodium acetate binds to negative charge of gDNA and neutralizes the charge. Neutralized gDNA can be precipitated in alcohol. |
| Gel electrophoresis is the process by which either proteins or ___A_______ can be separated based on ____B_______ when an electrical current is passed through a gel. | A. nucleic acid, B. size |
| Because gDNA has a net _A_____ charge, it will migrate from the ___B___ lead towards the __C____ lead. | A. negative, B. negative, C.positive |
| Generally, larger molecules migrate ______ than smaller molecules | slower |
| Resolution is affected by the ____________. | percentage of agarose |
| What percentage of agarose gel is used for gDNA extraction? Why? | 0.5%. Because gDNA is large molecule and lower concentration of agarose is used. |
| When you used higher voltage for gel electrophoresis, how will it be? | It will make separation faster, but resolution will be lower than low voltage. |
| What is the resolution limit of 0.5% gel? | 24kb |
| What is used for DNA ladder for the experiment(gDNA extraction) in the lab? | Lambda DNA which is uncut DNA of bacteriophage |
| What is added to gDNA sample to make visible? | loading dye |
| What consists of loading dye? | Bromophenol blue, sucrose, and EDTA |
| What is good points for SyBR Gold? | It is safer the ethidium bromide and more sensitive than SyBR Green. |
| Is gel electrophoresis is qualitative or quantitative? | qualitative |
| What kind of properties of gDNA sample can be assessed by spectrophotometry? | DNA purity, DNA yield, DNA concentration |
| In short, explain the Beer-Lambert Law. | absorbance and concentration is linearly proportional. |
| Spectrophotometry utilized the infrared to the ultraviolet portion of the ________________ to measure either the absorbance or transmittance of a substance for quantitative analysis. | electromagnetic spectrum |
| What wavelength indicates peptide bonds? | 230nm |
| What wavelength indicates nucleic acids? | 260nm |
| What wavelength indicates protein, based on the presence of aromatic amino acids? | 280nm |
| What wavelength indicates background? | 320nM |
| How dsDNA concentration can be calculated? | (A260-A320)*DF*0.05ug/uL |
| How protein contamination can be calculated? what is acceptable range? | (A230-A321)/(A260-A320) acceptable range is 0.4-0.6 |
| How Purity of DNA can be calculated? What is acceptable range? | (A260-A320)/(A280-A320) acceptable range is 1.6-2.0 |
| How the total volume can be calculated? | TE added-spec volume |
| For NanoDrop, protein contamination is calculated by different way. How is it calculated by NanoDrop? and What is the acceptable range? | A260/A230 acceptable range is 2.0-2.2 |
| For what part of DNA extraction Triton x100 was used? | It was used to lyse RBCs. |
| For what part of DNA extraction SDS was used? | It was used to lyse WBCs. |
Created by:
hiroko lucky
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