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58 Cards in this Set
- Front
- Back
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PCR stands for : _____
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Polymerase chain reaction
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DNA replication starts with the ____ defined by a specific _____
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replication origin
nucleotide sequence |
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DNA polymerase requires a ____ to begin copying
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RNA primer
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The RNA primer allows the ____ OH to be used by DNA polymerase
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3'
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Two Y-shaped replication forks are moving in the _____ directions during DNA replication
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opposite
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1: DNA polymerase adds to new RNA primer to start new DNA fragment
2: Nick sealed by DNA ligase to join new DNA fragment to the growing chain |
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Your body uses an RNa primer, in the lab we use a ____ primer
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DNA
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Doing this cuts out the step of ______
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removing the primer
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Both strands of DNA have to be replicated in the ____ direction
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5'--->3'
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DNA synthesis of the lagging strand is ______ and works in a ____ fashion
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discontinuous
backwards |
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The Klenow fragment aka
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DNA polymerase 1
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The Klenow fragment lacks _____ activity so it tends to copy DNA more than degrade it
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Nuclease (5-->3)
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Klenow denatures _____
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easily
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In repair replication a ___ primer was used and ____
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single
polymerase |
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Kary Mullis created ______
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Polymerase Chain Reaction
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You (can/cannot) violate a patent by 1. Extracting DNA from a species and copying it 2. Having purified Thermus aquaticus DNA polyermase 3. Thermocyclers/tubes
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can
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The only way you can use PCR products and process is if the experimental use is meant for __________
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curiosity or philosophical inquiry
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PCR will amplify a ______
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single target sequence
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PCR's use of _____ make it an exponential procedure
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two primers
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The temperature at which primers will anneal is ______
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variable
depending on the primers |
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1. Denature DNA
2. Anneal Primers 3. Extend Primers |
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The exponential growth of the ____ product is 2^n
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short
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The ____ products have linear growth
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long
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A limit will be approached as _____, _____, _____, _____, _____
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DNA begins to anneal to eachother because there is more DNA than enzymes
Loss of enzyme activity Limiting concentation of enzyme: Not enough enzyme to extend all the primers Background amplification If the sites are too far apart (>10 kb) |
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Polymerase cannot function without a _____ buffer
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magnesium
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Must have a ___ and ____ primer
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forward and reverse
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The gene with the "ATG" is called the ____ strand
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Watson
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The strand with the reverse complement (of ATG) : TAC is the _____ strand
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Crick
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The forward primer will have an ____ and it identical to whatever your gene sequence is
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ATG
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Forward primer will bind to the ____ strand
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Crick
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The primer that binds to the Watson strand has to be designed from the _____ strand
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Crick
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Watson goes _____
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5'--->3'
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Crick goes _____
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3'--->5'
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Oligonucleotides (primers) have greater than 50% _____ content
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G-C
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Primers are generally placed from _____ a part
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150-1000
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Do not want primers that can link back onto itself, called ______
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hairpins
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Do not want primers that can complement each other that can create _____ leading to amplification of the primers instead of the PCR product
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primer dimers
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The ____ can contain manipulations used for restriction enzyme digestion, his-tags...
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5' overhangs
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IT is important to know the ____ of your primer for the annealing stp
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Melting temperature (Tm)
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Tm= _______
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(A+T)x2 + (G+C)x4
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____ pull a part easier so have a lower Tm
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A&T
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To get 100% primer bound to target you take your lowest calculated Tm and ______
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reduce it by 5 degrees
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The primers must be pointed _____ and on ____ strands
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towards on another
opposite |
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The forward primer will bind on the Crick strand to the ___ codon
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anti-start
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The reverse primer will bind to the ____ strand and amplify towards the ____ codon
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template
start |
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The forward primer will always be the same as the _____ at about 20 nucleotides long
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template strand containing Gene of interest
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The reverse primer can be created by taking the ____ nucleotide complementing it and writing it in the ___ direction and keep going left
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first
forward AAGGCGCAAGTAG (G.O.I) CTACTTGCGCCTT (Reverse primer) |
Can add an EcoR1 cut site for _____; Can add a his-tag for _____ and if you have a his-tag you must also have a _____; The GC is present at the beginning of the 5' end because _____
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restriction enzymes to cut in order to place the GOI into a plasmid
washing the sample over a column and purifying your GOI (affinity chromatography) Start codon It is a flanking base needed by restriction enzymes to cut at the restriction point |
On the reverse primer you must delete the ___ and move it to after the _____; The Xho1 is _____; Add GC at the end to ___
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stop codon, His-tags (so that it will stop making them)
another cut site to a different restriction enzyme accommodate the restriction enzymes |
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The manipulations of overhangs will always be at the ____ end
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5'
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The manipulations of overhangs will always be at the ____ end
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5'
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The primers designed with EcoR1 and Xho1 will bind to the _____; the product formed will be ____
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Gene of interest on the cDNA library
smaller, only the GOI |
You are then able to place your product into a ___ using the cut sites and transform the plasmid into bacteria and express the GOI in the cells
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plasmid
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In overlap Extension PCR cloning you do all the work on the ____
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plasmid
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In ligation independent cloning you do not use ____ or _____; The ligation will occur through _____
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restriction enzymes, ligation
E. coli's DNA ligase |
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Piecing genes together on gene without doing digestion, ligation or any recombination
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Circular Polymerase Extension cloning
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In long PCR you ___ the GOI creating more than one gene product, you then _____ to get one product
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break up
digest and ligate |
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In oligonucleotide sequencing you ____ the sequence and use yeast to join the fragments back together
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break up
(<100k bp) |
