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108 Cards in this Set
- Front
- Back
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What is a plasimid?
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A plasimis is dbl stranded circular DNA.
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What are specific DNA cutting enzymes?
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restriction endonucleases
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What does the length of the recognition sequence of a restriction endonuclease determine?
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The number of cuts that will be made in the DNA. Tha larger the recognition sequence, the fewer cuts will be made. (The prob = (1/4)^num of b.p, in sequence.
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Draw a 3'-hydroxyl extension cut.
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P P P P P OH
S S S S S C T G C A ' ' ' ' ' ' A C G T C S S S S S S HO P P P P P P |
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Draw a 5'phosphate extension
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see note
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Draw a blunt end clevage
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see book
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What is the class of endonucleases used most often and why?
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Type II becuase there are seperate restriction and methylating systems, they have a relativly simple structure, and all you need is Mg2++ to make it work
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Where are restriction enzymes used?
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cloning DNA, Mapping, RFLPs analysis
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What is star activity?
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The optimal way to run a reaction for a particular enzyme.
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What are isoschizomers?
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Restriction endonucleases that overlap in terms of recognition sequences.
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How do you calculate the fragment size of a particular restriction endonuclease?
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4^n
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What do you need to do in order to recombine DNA?
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cut the source DNA and the vecotr DNA with the same restriction endonuclease, mix and aneal
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What is a nick?
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A break in the phosphdiester bonf in one strnd of the dbl stranded DNA
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What is annealing?
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Comming together of base pairs via hydrogen bonds
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What does ligase do?
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Gets rid of nicks
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How do blunt ends reaneal?
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It depends on the probibility of the two ends comming together and being ligated. There are no H bonds in blunt ends
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How would you prevent plasmid DNA from reanealing with itself?
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Use alkaline phosphatase to remove the 5' phosphate. You do it to the plasmid and not the source DNA because target DNA is too short
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What are the 3 main types of polymerases?
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Polymerase I, Klenow Fragment, Reverse transcriptase
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What are the main charactaristics of DNA Polymerase I?
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It needs a template (a piece of dbl sranded DNA) eg primer
It attacks nicks (moves them along the DNA) It has dual activity as a polymerase and a exonuclease |
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What are the main characteristics of Klenow fragments?
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Has the synthesis activity of polymerase I without the degrading activity
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WHat are the main characteristics of reverse transcriptase?
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RNA dependant DNA polymerase
it needs a template |
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What direction is synthesis?
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5'-->3'
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What is a uniques feature of mRNA? Why is it imporatnt?
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mRNA always has a poly A tail. This is important because it can be easily separated from other RNA with the use of a poly T chain in a column.
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Why is mRNA a good place to start when getting source DNA?
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mRNA is always protein coding
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What does polynucleotide kinase do and why would it be useful?
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Polynucleotide kinase adds a 5' phosphate. I don't know.
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Describe the process of using reverse transcriptase to get DNA?
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You need an mRNA. The mRNA has a poly A tail ( at 3' end). Attatch an oligo(dT)primer to the poly a tail. Use reverser transcriptase to synthesize the first line of cDNA. Nick the mRNA template with RNase H (so it can be removed).Use DNA polymerase to synthesize second strand. Use DNA ligase to seal up nick with the RNA primer (at the 5' end)
OR the better way to prevent synthesis of incomplete strande : 1. use oligo(dT)primer 2. Use reverse transcriptase to synthesize first DNA strand. 3. The reverse transcriptase will loop (hopefully at the end of your mRNA strand) and create a primer for your second DNA strand 4. Use Klenow to synthesize second DNA strand 5. remove mRNA strand with RNase H; s1 nuclease to degrade single stranded DNA |
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What are terminal transferases and what do they do?
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Terminal transferases join blunt ended DNA and are used for homoplymer tailing. They as 1 or more deoxynucleotides to the 3' terminus.
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Why would you lable DNA?
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so you can do a Southern Blot
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How would you label DNA?
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with lables single stranded probes. Hypridize these probes with single stranded DNA
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What are random heximers?
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6 base primers asses to single stranded DNA. When Klenow and labled dNTPs are added it extends off of the dbl strand and puts in the labled dNTP
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What is the main function of vectors?
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They are carriers for DNA and allow host cells to replicate the vector. The can also provide methods of SELCTING transformed cells.
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WHat is the ORI? Why is it important?
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The ORI (origin of replication)is where translation takes place. You DO NOT want to cut the plasmid there. The host cell must recognize the ORI fo the plasmid in order for them to be compatible.
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Why is the pUC 19 plasmid a dual screening vector?
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It has two selectable markers: and Ampacilline resistance gene, a LacI gene, and a Lac z gene with a multiple cloning site inside of it. This allows dual screening: does the bacteris have the plasmid= is it resistant to ampicillin and does the bacteria with the plasmid have a DNA insert = no color because lactose metabolism is disrupted
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Why would you use a phage vector instead of a plasmid?
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can hold larger pieces od DNA
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What are the 2 classes of viruses?
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Head and tail, which will eventually lyse the host cell and filamentous which does not kill the host cell. It makes copies and exports itself.
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What is a filamentous virus useful for
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DNA sequencing (The DNA is single stranded and circular once in the host)
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What are COS?
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cohesive ends of a viral vector
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Where do you put an insert into a bacteriophage?
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You can remove genes that are needed for the lysogenic life cycle (which is unecessary for cloning) and insert there.
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What is a shuttle vector?
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a vector for multiple host cells
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What is necessary to have in a shuttle vector?
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An ORI that is complatible for both organisms and selectable markers for both organisms
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How large (APPX) is a viral insert?
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15-20kbp (or 20,000 bp)
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How large is a plasmid insert?
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4,000 bp
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How large is a cosmid insert?
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40,000 bp
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How large is a YAC insert?
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400,000 bp
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What is a linker?
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a short DNA sequence that can be added at the end of a gene of interest.
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Why would you add a linker?
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If you want to put an insert into a cloning vector and you need a certain endonuclease end to do it, and the cDNA does not have the endonuclease site in the right place, you need to add it at the ends as a linker. Then the restric. endnuclease has a place to cut. Sometimes it is necessary to methylate the cDNA before this step to prevent the restriction enzyme from cutting within the target DNA.
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What are codmid vectors?
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The are round like pladsmids but have a COS site and can be packaged into viruses. When it infects a host cell the cell recognizes the cosmid as a plasmid.
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What is auxotropic selection in yeast?
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In yeast it is using a mutant that cannot synthesize a certain essencial amino acid as a marker. If you add the gene in plasimis with transformed cells the yeast can grow.
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What does detection screening determine?
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If the transformed bacteria cell has the right DNA
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How do you perform detection screening?
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1. transfer colonies onto membrane
2. degrade cell, purify DNA, bind single stranded DNA to membrane 3. Probe with labled DNA (labels attatched to DNA with the correct sequence) 4. x-ray for positive hybridization 5. go back to master plate and retrieve colonies |
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Why would you want to screen at the protein level?
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because the DNA could be in reverse orientation.
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How do you screen at the protein level?
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Use anitbodies:
1. transfer cells to membrane 2. lyse cells and transfer proteins to matrix 3. treat matrix with primary antibody (wash away excess) 4. Treat matrix with secondary antibody (that binds to primary antibody and that has a color reaction)(wash away excess) 5. examine for color reaction and return to master plate |
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What is a method of screening for knocked out pathways?
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complementation
(only transformed cells can grow on minimal medium) |
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How do you make sure you have a representation of genes in your library?
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calculate the number of clones needed:
N = ln(1-P)/ ln (1- 1/n) N= number of clones P= probibility (you set the probibility) n= size of genome/size of fragments Example:95% probibility of including all genes in a random human genomic library of 20 kb fragment size is... N= ln (1- 0.95)/ln(1-1/1.4 x 10^5) N= 4.2 x 10^5 |
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What is the target of a sothern blot? What is the probe?
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DNA is the target
oligonucleotide it the probe |
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What is an oligonucleotide?
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A short sequence of nucleotides.
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What is the purpose of biotinylation?
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The biotin marker at the end of an mRNA in the cDNA library construction process will allow binding with a streptavidin-coated magnetic bead.
Also, when making a cDNA library, the biotinilated mRNA strand that does not have a complete complementary DNA strant attatched to it has a portion of single stranded RNA between the biotin marker and the incomplete DNA strand. When RNase I comes along it will degrade this portion and the biotim marker will have no mRNA attatched to it. Therefore, all of the magnets with ANY mRNA must also have a complete DNA strand. |
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What does RNase I do?
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degrades single stranded RNA
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Why would you use a guessomer?
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If you know a proteins amino acid sequence but not the DNA or mRNA sequence
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What is a guessomer?
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A possible DNA sequence generated from an amino acid sequence.
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How can you narrow down the DNA sequence possibilities when building one using guessomers from an amino acid sequence?
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Check the codon preference for the organism
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What is the target of a northern blot? What is the probe?
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target is RNA
probe is oligonucleotide |
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What are the methods for transforming cells?
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1. salts and temperature (give heat shock to cells, cells will take in DNA, allow recovery)
2. Electroporation (suspend cell in liquid, apply electricity, holes form in membrane, cell takes up DNA, let cells recover) 3. Liposomes (mix DNA with lipid solution to form liposomes, expose host cell to liposomes, liposomes fuse with membrane, DNA is taken up) 4. Balistic Bombardment 5. Viral systems |
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Explain transformation through salt and temperature method
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give heat shock to cells, cells will take in DNA, allow recovery
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Explain transformation via electroporation
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suspend cell in liquid, apply electricity, holes form in membrane, cell takes up DNA, let cells recover
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Explain transformation via liposomes
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mix DNA with lipid solution to form liposomes, expose host cell to liposomes, liposomes fuse with membrane, DNA is taken up
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What is the role of the promoter in prokaryotic cells?
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a promotor controls gene activity, when and how much of a gene is made
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What is important to remember when working with prokaryotic systems with promoters?
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A promotor must be compatible with the host polymerase
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How can you contol that host cells make the maximum amount of protein at the same time?
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If the gene has a regulatable promotor you can control translation of a gene with chemicals or temperature
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When sequencing DNA what woulf be the strategy for cloning DNA?
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Start with the larget cloning vectors (YACS) and move on to smaller ones (plasmids)
((start with large chunks od DNA and then go down to single genes)) |
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What is a YAC? What are some of its features?
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A YAC is a Yeast Artificial Chromosome. It is linear and ends in a telomere. It can take clones over 100kb long. It has selectable markers for bacteria and yeast. (IT can be replicated in mass in an ecoli and then yeast)
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What is the purpose of chromosome walking?
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fpr full sequencing of a chromosome
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HOw does chromosome walking work?
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clone and sequence a 500-800bp region at a time. Overlap the ends and use it as a primer for next 500-800 bp.
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What is the down side to chromosome walking?
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It is a slow process becuase you must finish the 1st sequence before moving on
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What does the T-DNA region of a Ti plasmid code for?
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The transfer DNA that will be inserted into the plant. Usually it contains a left and right border, auxin inducing genes, cytokinin inducing genes, opine producing genes
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How is a Ti plasmid usually activated?
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The virulence region is activated when the plant gives chemical signals that it is wounded.
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What are the limits to native Ti vectors?
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hormones prevent maturation of plant
opines production suck energy from the plant and cause a lower yeild too large for cloning needs ori for Ecoli |
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What does the engineered version of the Ti plasmis have?
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-selectable maker (for antibiotic)
-ori added for ecoli -right boarder of T-DNA -MCS within the arms -lack or vir genes (on another plasmid) |
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Why is it benificial to use the Ti plasmid as a co-integrated cloning vector?
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you can grow small plasmids more effectively then recombine them
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What is PCR? What is it used for?
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PCR is the polymerase chain reaction. It is used to amplify DNA. (make lots of DNA fast)
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What are the steps of PCR?
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There are 3 in a cycle
1. denature to seperate DNA (94 degrees for 30 sec) 2. Add primers to DNA strands and aneal (30-65 degrees for 30 sec) 3. Polymerase synthesizes new DNA strands (extension) (65-75 degrees c for 2-5 min) REPEAT! |
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What kind of polymerase is used in PCR and why?
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Polymerases from a bacteria that lives in high temperatures is used (Taq) becuase it can work in high temperatures
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What is necesarry for PCR?
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1. two synthetic oligonucleotide primers that are complimentary to the regions on opposite strands that flank the target DNA sequence and that after annealing hace the 3' ends facing each other
2. a target sequence of DNA that lies between the primers 3. a thermostable DNA polymerase 4. the 4 deoxyribonucleotides |
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Why should you not run a PCR reaction too long? What is too long?
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After too many cyles contamination becomes a problem. (You are ampliffing junk as well as your DNA)
Stick to below 20 cycles |
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Why would you chose to clone using a vector as opposed to PCR?
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you need to know something about the DNA to use PCR (you need to make a primer)
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What is Tm? Why is it important?
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Tm is the melting temperature of DNA
Tm = 2(A+T) + 4(G+C) It is importnt because it determines the anealing temperature. The higher the anealling temperature the more specificity |
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How do you avoid nonspecific priming in PCR?
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hot start
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What is essencial when it comes to primer binding in PCR? what is not? What does that mean?
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It is essencial that the 3' end of the primer to bind very strongly because you need the DNA polymerase to bind to the brimer and to begin synthesis (5'--> 3')
The 5' end is not as important. This allows you to put primers, adaptors, sites for endonucleases etc. at the 5' end which you can use later. |
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What is wrong with your PCR if the primers and templates remain dissacociated? What do you do?
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The annealing temperature is too high. lower it!
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What is wrong with your PCR if there is mismatching hybridization of primers?
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The annealing temperature is too low. raise it
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What are the limitations of the Taq polymerase?
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- adds extra nucleotides (A) at the 3' end
- It needs a specialized cloning vector - It has a high error rate |
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Why does Taq have a high erroe rate?
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little exonuclease editing activity
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Why would the high error rate of Taq be useful?
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To induce random mutation
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What are nested primers? Why are they used?
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Nested primers are added to a PCR reaction to increase specificity. Sometimes primers can bind at undesired sequences. A nested primer will bind within the other primer only on the target sequence strand. Then the PCR reaction continues with the new nested primer and the undersired sequence is not amplified.
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What is the purpose of asymetrical PCR?
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to get a lot of copies of only one strand of DNA and not the other (for sequencing)
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How is asymetrical PCR done?
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only one primer is available so the strand without the primer is not amplifyed nearly as much.
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What are the benefits of Real time PCR?
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-quantitation
-log view (measure kinetics of amplicon accumulation in exponential phase) -direct readout (no gels) -the higher the start copy nember the sooner a significat increase in flourescence is detected |
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What are the florescence detection methods for real time PCR?
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-hydrolysis probes (TaqMan, molecular beacons, scorpions)
-Hybridizing probes (SYGR-green) -DNA-binding agents |
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How does the TaqMan probe work?
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the TaqMan has a flourescent dye at the 5' end of the probe and a quencher at th 3'
It anneals with an amplified region When an enzyme reached to probe the 5' nuclease activity displaces the probe. Gives off flurescence as quencer is seperated from dye |
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How does SYBR green work?
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It aneals to dbl stranded DNA and flouresces (glows only when DNA is dbl stranded)
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What are the benefits of Real time pCR?
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kinetic data (exponential phase) vs end point (plateau)
increase in flourescence directly related to # of amplicons Increased dynamic range of detections No post PCR processing Detects two fold change in starting DNA |
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What does a DMT (dimethyloxytritl) do in the chemical synthesis of DNA?
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It is attatched to the OH 5' end of a nucleoside to prevent the 5' hydroxyl group from reacting nonspecifically before the addition of a second nucleotide.
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What happends to the DMT?
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Before the second nucleotite is introduced the DMT is removed with trichloroacetic acid (TCA) and a 5'hydroyl is left
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What is the purpose of the capping reaction in the chemical synthesis of DNA?
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The 5' hydroxyl of unreacted nucleosided are capped to prevent them from reacting with the next nucleotide. If this step wasn't performed you would have lots of different DNA sequences of different lengths.
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What is the purpose of the oxidation reaction in the chemical synthesis of DNA?
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This strengthens the phosphodiester bond and makes it less susseptable to clevage under acidic or basic conditions
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Why would you need oligo synthesis?
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To make likers/adaptors
To make guessomers To make synthetic genes |
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What are the two ways in which you can make a gene syntheticaly?
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make individual strands that will base bair with eachother and aneal
or synthesize single strands that overlap but have gaps use polymerase to fill in gaps |
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What is the reason you would use a dideoxynuclotide instead of a standard deoxy?
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a dideoxy cant form a phosphodiester bong with the next nucleotide. It stops the synthesis of DNA. It is used for DNA sequencing. You can find out where the DNA ends (at what base pair at what length)
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