Term
| Gel electrophoresis seperates via |
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Definition
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Term
| Which side of gel does sample usually go into? |
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Definition
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Term
| Which samples travel furtherst/ and slowerst |
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Definition
Large= slow, travel the least
Small= fast, travel the most |
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Term
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Definition
| seperates chunks of dna via size |
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Term
| 1st step of southern blot- |
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Definition
| suspect/sample dna is cleaved into small pieces via endonucleases hydrolyzing the phosphodiester bonds. |
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Term
| Marker is ran in the gel to- |
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Definition
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Term
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Definition
| probe for specific bands by incubating the gel with probe a dna molecule with specific bp complimentary specific seq. this creates dsDNA. probe is labelled with color so you can see withch bands are a match. |
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Term
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Definition
| same as southern blot but for RNA. |
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Term
| technique to sequence the DNA= |
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Definition
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Term
| sanger method starts with |
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Definition
| ss dna sample for the template |
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Term
| sanger method with the template it is- |
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Definition
| incubated with large amount of dNTPs primer, dna polymerase, buffer, and small amount of modified dideoxyNT |
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Term
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Definition
| are analogs to A,C,T, or G. are missing the 3' OH group and are colored |
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Term
| Sanger method elongates strand |
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Definition
| until the di deoxyNT is added then elongation stops and there is a colored label on the 5' end of the strand |
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Term
| why does elongation stop with the di deoxyNT? |
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Definition
| cant elongate bc there is no 3' OH group no oxygen to do a nucleophillic attack on the next NT triphosphate to keep creating the backbone. |
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Term
| After the synthesized dna fragments of different lengths are made in the sanger method what happens? |
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Definition
| denatured into ss dna again and read in the gel by length. |
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Term
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Definition
| 4 tubes with only 1 analong ACGT each and the primer was labelled instead. |
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Term
| the colored modified base will be at the -- end of new fragment? |
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Definition
| 3' end. DNA is synthesized 5'->3' |
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Term
| sanger method needs -- before it can start |
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Definition
| must figure out the complimentary pairing for the primer first |
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Term
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Definition
| amplify dna to a larger concentration of dna with same bp seq |
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Term
| What do you put into the pcr thermocycler: |
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Definition
2 primers (start with the ds dna)
buffer
4 DNTP |
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Term
| in pcr the primer must be |
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Definition
| complimentary to the 5' end of the piece of the template dna you want amplified |
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Term
| steps of pcr and temps/times |
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Definition
denature 95 for 12min
anneal 60 for 1 min
extending 70 for 1.5min
repeat |
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Term
| first step pcr denaturing- |
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Definition
| heat depends on CG vs AT content needs to seperate the ds dna to ss by breaking hydrogen bonds |
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Term
| 2nd step of pcr annealing |
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Definition
| primers form compliments with template. primer can form h bonds at 60 degres for bp. |
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Term
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Definition
| elongation of primers at 70 degress dna polymerase adds DNTS in the 5'->3' direction |
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Term
amplification totals=
20 cycles= --dna |
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Definition
2^cycle #
20 cycles= DNA 1million copies |
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Term
| after the last pcr cycle- |
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Definition
| quench at 4 degrees and ds DNA will reform |
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Term
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Definition
| Taq polymerase protein stable at high temp. not used up bc it is an enzyme-> dont have to add more after each cycle. |
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Term
| restriction enzymes/ restriction endonucleases- |
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Definition
| enzymes that hydrolyze phosphodiester bonds at specific sequences called palindrome |
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Term
| bacteria protect there dna from restriction enzymes by |
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Definition
| methylatning groups to some a or C groups at their restriction sites |
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Term
| restriction endonucleases cleave |
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Definition
| both strands (both phosphodiester bonds) in the middle of dna |
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Term
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Definition
| when read 5' ->3' the sequence is identical |
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Term
| Taq1 polymerase recognizes |
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Definition
4 base and cuts in stick ends
TCGA. cuts at T C bond |
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Term
| Hae3 polymerase recognizes |
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Definition
4 base and cuts blunt ends
GGCC cuts between C and G |
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Term
| EcoR1 hydrolyzes at -- and ends up with -- |
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Definition
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Term
| restriction enzymes are named |
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Definition
1st letter= genus
2nd two= species
4th- strain
numeral= order it was found in that bacteria |
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Term
| average freq of cuts for a restriction enzyme in dna? |
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Definition
4^n
4 for each ACTG
n= length of palindrome it recognizes |
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Term
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Definition
| 4096 bp; bigger fragments |
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Term
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Definition
| 256 bp; smaller fragments |
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