Molecular bio lab test #1 Flashcards _ Quizlet

3/25/24, 11:13 AM Molecular bio lab test #1 Flashcards | Quizlet https://quizlet.com/37076198/molecular-bio-lab-test-1-flash-cards/?funnelUUID=4d8faa94-c387-46bf-b2f3-7cd75b6fd9c4 1/5 Molecular bio lab test #1 Leave the first rating Students also viewed Terms in this set (73) Others also viewed these textbooks Search for a textbook or question Hole's Human Anatomy and Physiology 13th Edition • ISBN: 9780073378275 (15 more) David N. Shier, Jackie L. Butler, Ricki Lewis 1,458 solutions Bio 20 terms sabrivillaa09 Preview Genomics Test 3 72 terms AlyssaM1502 Preview GMOs & CRISPR Basics 12 terms evecaselden Preview Bio 111 Lab 50 terms Alexa_G Purpose of electrophoresis To separate, identify, and purify DNA, RNA, or proteins Parameters used to separate molecules in agarose gel the agarose gel has the capability of separating by size and charge but since all DNA has a negative charge we separated based on size How does the agarose concentration affect the seperation of molecules the higher the concentration of agarose th better the separation of smaller DNA fragments What is the normal range of agarose gels that labs tend to use? 0.6%, 0.8%, and 1.0% In electrophoresis the DNA runs toward which pole and what is its name? it runs toward the anode which red because DNA is neg and the anode is the positive pole In electrophoresis which pole is the negative pole and what is it called? Black pole is negative and it is called the cathode because they are named for what they attract Name the factors that influence resolution of DNA in an agarose gel agarose concentration, the buffer used, the voltage used and salt concentration How does the agarose concentration affect the resolution of DNA in an agarose gel electrophoresis The higher the concentration of the agarose gel the smaller the fragments we can separate What are the two types of buffer solution used in electrophoresis that may affect DNA resolution and describe each? TBE buffer used for plasmids, separates DNA 50bp-10,000bp TAE buffer used for different sized DNA, most common type, used for genomic DNA, separates DNA > 10,000 bp Molecular bio lab test #1 Study

3/25/24, 11:13 AM Molecular bio lab test #1 Flashcards | Quizlet https://quizlet.com/37076198/molecular-bio-lab-test-1-flash-cards/?funnelUUID=4d8faa94-c387-46bf-b2f3-7cd75b6fd9c4 2/5 What does the buffer do in the electrophoresis Buffer assists with migration of DNA prevents accumulation of H+ and OH- How does the voltage affect resolution in electrophoresis and when would you use either one? Higher voltage is needed to resolve smaller dna (5 volts/ cm=125volts); Lower voltage is used for larger DNA 10-12kb (1-2volts/cm =70volts) how does the salt concentration affect the resolution of DNA on an agarose gel in electrophoresis use low salt buffer for DNA suspension( small amounts of salt keep the DNA stable but too much can denature) Purpose of gel loading buffer? To increase density of sample(glycerol) and to add color to visualize the sample Which two dyes were used in the gel loading buffer in lab 2 electrophoresis bromophenol blue and xylene cyanol describe bromophenol blue it is in the gel loading dye in electrophoresis lab, it is the leading dye, it moves faster (300bp) describe xylene cyanol it is a component in the gel loading dye in electrophoresis lab, it is the lagging dye so it moves lower (4000bp) bromophenol blue moves _____ fold faster than xylene cyanol 2.2 Name all the visualizing agents that may be used for DNA in a agarose gel methylene blue, Ethidium Bromide,SYBR green, GelStar stain describe Methylene blue and its disadvantages/advantages adv- non-toxic disadva- low sensitivity, need >40ng of products, can take a long time describe Ethidium Bromide and its disadv/adv Adv- sensitive, detects >1ng DNA, inexpensive Disadv- strong mutagen, possible carcinogen, need UV light How does Ethidium Bromide work it intercalates between stacked bases of DNA, so in otherwords it wedges itself between Ts and As describe the disadv/adv of the SYBR green stain adv- very sensitive (>60pg DNA), safer than EtBr disadv- expensive describe the disadv/adv of the GelStar stain adv- 5-10x more sensitive than EtBr disadv- expensive What are the disadvantage/advantages of using polyacrylamide gel advantages- Better resolution of smaller DNA (5-500bp), can separate DNA with only one base pair difference in size Disadvantages- More difficult to prepare, uses vertical gel apparatus What is the purpose of using a "marker" DNA To have something to compare your unknowns against, With a marker we will know the marker band fragment sizes are and then can make a graph utilizing the distance migrated vs size to then compare unknown bands and estimate unkown fragment size What is the source of lambda DNA it comes from a bacteriophage lambda, E. coli is the host for this particular bacteriophage which restriction enzymes were used in lab 3 HindIII and EcoRI why were the restriction reaction tubes incubated at 37 degrees in lab 3 (restriction digest and electrophoresis of lambda DNA) because 37 is the optimum temp at which these particular enzymes work best at so we placed our tubes at 37 degrees to aid the RE in cutting What are restriction enzymes and where are they found in nature Restriction enzymes are scissors that cut both strands of DNA, found in bacteria which make RE to fight viruses or bacteriophages Why do bacterial cells produce restriction enzymes to cut up bacteriophage DNA that enters the bacterial cell Molecular bio lab test #1

3/25/24, 11:13 AM Molecular bio lab test #1 Flashcards | Quizlet https://quizlet.com/37076198/molecular-bio-lab-test-1-flash-cards/?funnelUUID=4d8faa94-c387-46bf-b2f3-7cd75b6fd9c4 4/5 explain how you would set up the transfer apparatus in a transfer pan place an upside down gel chamber, then place a long piece of whattman paper to act as a wick, next place the gel, then the nylon membrane, several more pieces of gel sized whattman paper, then a stack of 6 inches of paper towels held down by a medium glove box (used as a weight) saran wrap all four sides what is the purpose of using a Pos. charged nylon membrane for the transfer of DNA (lab 4 southern blot) because under conditions such as ours ( a pos charged membrane and alkaline transfer buffer) the transferred DNA bonds covalently to membrane during transfer which reduces possible loss of DNA by leaching through membrane during blotting what is the purpose of the paper wick in the transfer apparatus to move transfer buffer from dish through gel and into membrane and paper stack which will cause the DNA to move out of gel and onto membrane by capillary action what is the purpose of the saran wrap in the transfer apparatus to prevent evaporation, to prevent anything from falling into or hanging in solution which would bypass the wick What is the purpose of the UV crossilinker? and how does it work? To fix DNA to the membrane, it causes formation of covalent bonds between thyamine in the DNA and the amino group of the nylon membrane Explain PCR 1. heat to 94-96 C to denature target DNA (separate into single strands) 2.Temp lowered to 50-65 C to allow primers to anneal (base pair) to complementary sequences (bracketing the target DNA) 3. Temp raised to 72 to allow Taq polymerase to attach to each priming site and synthesize an new strand THEN CYCLE REPEATED OVER AND OVER what components do you need for successful PCR DNA template, primers, dNTPS, a polymerase, pcr mix what were the three temps that the thermocycler ran at in our PCR experiment 94,55,72 how many cycles do you usually run in PCR 25-30 in PCR "n" cycles will result in how many strands 2^n strands what is Taq Polymerase a thermostable DNA polymerase (an enzyme that synthesizes DNA from nucleotides) names after Therus aquaticus, it is used in PCR because it can withstand high temps in the thermocycler What was the name of the spin column we used? Does it retain agarose or DNA? GenElute , agarose What is the purpose of adding ammonium acetate and isopropanol and how do they work together? the ammonium acetate is positively charged neutrilizes the neg charge of the phosphate groups of DNA, so it makes the DNA less hydrophillic and therefore less water soluable, the isopropanol aids the ammonium acetate and makes it more effective by changing the electrostatic attratction between the ammonium (salt) and the phospate groups of DNA and increases the attraction between the ammonium and the phosphate groups of DNA In lab 6 Probe labeling why was the DNA boiled prior to labeling? to denature the DNA to make single stranded, What would happen if the DNA was not single stranded in the probe experiment if not single stranded the hexanonulceotide primers couldnt bind what are hexanucleotides contains 6 nucleotides, single strandes DNA of random sequence, this acts as a primer and provides a starting point for DNA synthesis what are dNTPs as they are used in lab 6 Probe labeling this contains loose nucleotides, some of which are labeled. these loose nucleotides are taken as building blocks Molecular bio lab test #1

3/25/24, 11:13 AM Molecular bio lab test #1 Flashcards | Quizlet https://quizlet.com/37076198/molecular-bio-lab-test-1-flash-cards/?funnelUUID=4d8faa94-c387-46bf-b2f3-7cd75b6fd9c4 5/5 what is the Klenow enzyme a frag of E coli DNA polymerase I. The enzyme synthesizes DNA from nucleotide precursors. IF the nucleotide contains dig then it will be labeled what is Digoxygenin and its purpose Dig is a primary antibody used to label DNA, comes fro a steroid isolated from digitalis plant or foxglove; a secondary antibody must be conjugated to it for visualization why was EDTA added to the reaction tubes after incubation in lab 6 probe labeling EDTA is a chelator so it will bind with the magnesium ions which the Klenow enzyme requires to function so EDTA hinders the Klenow enzyme from working thus stopping the reaction What is the purpose of blocking buffer to prevent non-specific binding what was the purpose of control DNA used during testing of labeled probe to be able to determine concentration of our original sample probe Explain how anti-dig is used to detect labeled probe anti-dig only binds to dig labeled probes so the dig is primary ab and the anti-dig is secondary ab what is the color substrate used to detect labeled probe NBT/BCIP in 10 ml of water purpose of prehybridization buffer to prevent non-specific binding of probe DNA to membrane what are the reagents found in the prehybridization buffer 20xSSC, DI water, Skim milk (powdered), 10% N-laurylsarcosine, 10% SDS what is the purpose of 20xSSC in the hybridization solution salt (SSC) in prehybridization/hybridization mix allows matching DNA to hybridize to one another Why was labeled probe boiled prior to adding hybridization solution to make probe single stranded so that it may bind to DNA on membrane explain the purpose of wash and blocking buffers in lab 8 detecting lambda dna on membrane blocking buffer- covers any unbound parts of membrane to reduce non specific binding washing buffer- washes off loosely bound things from previous step common causes for lack of signal( bands ) on membrane probe was not properly labeled, probe not boiled prior to make single stranded, DNA didnt transfer to membrane common causes for high background color on membrane reaction allowed to go to long, blocking solution not made properly, blocking reaction not preformed correctly, membrane was not kept in dark, dried membrane was exposed to a bright light, too much anti-dig was used explain the reaction between the color substrate and the alkaline phosphatase BCIP is oxidized by the alkaline phosphatase to indigo by release of a phosphate group (dephosphorylation) and NBT in parallel is reduced to diformazan. The reaction products form a water insoluable dark blue to brownish precipiate, depending on type of membrane Molecular bio lab test #1