Restriction Enzyme

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Restriction Enzyme
Digestion
Store in -20 C
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Restriction Enzyme
Buffer NaCl or KCl, TrisHCl, MgCl2,
DTT Different salt varied activity Buffer
supplied with enzyme for 100 activity
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Restriction Enzyme
Buffer Universal buffer for double or triple
digestion some activity reduced Check manual
from company if different buffer to be used
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Restriction Enzyme
Methylation Modification commonly found in DNA
of bacteria, eukaryote and their virus m6A,
m5C, m4C, hm5C
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Restriction Enzyme
Temperature In general 37 C 67, 25 or 50 C also
found
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Restriction Enzyme
Enzyme inactivation Heating 65 C for 15
min Extraction with phenol/chloroform Addition
of EDTA
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Restriction Enzyme
Star activity Cleave nucleic acid
nonspecifically Factors Non optimal
pH Substitution of Co2, Mn2, Zn2 for Mg2
Increase enzyme concentration Reduce salt
concentration High glycerol (prevent
freezing) Organic solvent higher than 1
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Electrophoresis
Analytical method for purification / isolation
separation / fractionation identification Elect
rical field Simple and Rapid Gel medium / Buffer
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Electrophoresis
Agarose / Polyacrylamide gel Detection by
staining Ethidium bromide (UV) Acridine orange
(UV ss-orange / ds-green) Silver
staining Brilliant blue or Methylene blue
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Range of Separation
Agarose in 1x TBE Linear dsDNA (kb) AcrylamideT in 1xTBE Linear dsDNA (bp)
0.3 0.6 0.7 0.9 1.2 1.5 2.0 5 - 60 1 - 20 0.8 - 10 0.5 - 7 0.4 - 6 0.2 - 3 0.1 - 2 3.5 5.0 8.0 12.0 15.0 20.0 100 - 1000 75 - 500 50 - 400 35 - 250 20 - 150 5 - 100
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Electrophoresis
Agarose gel Linear polymer of D- and
L-galactose Quality varies from batch to
batch Separate few hundreds to about 20 kbp Low
resolving power Run in horizontal configuration
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Electrophoresis
Agarose gel LMP agarose to be run at 4 C TAE
(high MW) or TBE / TPE (low MW) Gel loading
dye bromophenol blue, xylene cyanol
FF sucrose, ficoll, glycerol
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Agarose Gel Electrophoresis
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Agarose Gel Electrophoresis
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Polyacrylamide Gel Electrophoresis
For protein analysis For smaller DNA
fragments High resolving power Run in vertical
configuration
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Polyacrylamide Gel Electrophoresis
Acrylamide and Bis-acrylamide APS and TEMED
without oxygen Denaturing PAG with
Urea/Formamide for ssDNA
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PAGE
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PAGE
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PAGE
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Polymerase Chain Reaction
Temperature Time
Denaturation Annealing Extension
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Polymerase Chain Reaction
Components
Thermostable DNA polymerase Template
Primer Substrate Buffer
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Polymerase Chain Reaction
Template small amount (1 ng 1 ug) free from
contamination Primer specific to target /
dimer nt length (Tm) / 1-2 primer (s) / GC
content modification mutation / RE site 0.2-1
uM
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Polymerase Chain Reaction
dNTPs 50-200 uM each recommended MgCl2 1.5 mM
recommended effect on primer annealing / Pol
activity Gelatin /BSA enzyme stability DMSO
better denaturation of long target
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Polymerase Chain Reaction
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Polymerase Chain Reaction
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Polymerase Chain Reaction
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Thermal Cycler
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Polymerase Chain Reaction
Hot start PCR enzyme activity blocked
during reaction setup enhance specificity /
sensitivity increase target yield by wax,
chemical or enzyme Ab
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Polymerase Chain Reaction
Nested PCR Product of previous reaction used
as template for next reaction New sets of
primers correspond to sequences internal to
the previous set
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Polymerase Chain Reaction
Multiplex PCR combined primer sets amplify more
than 1 target in 1 tube
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Polymerase Chain Reaction
Real time PCR quantify amplified
products comparative assay of initial
templates monitor increased fluorescence
signal during cycles (not end point)
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Polymerase Chain Reaction
Touch-down / Step-down PCR annealing temp
progressively reduced from above Tm to below
Tm specific target amplified at high stringency
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Polymerase Chain Reaction
Degenerate PCR sequence of target not known
exactly eg. to find new gene or gene
family back translate from protein mixed
primers with wobbles
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Polymerase Chain Reaction
Degenerate PCR Trp Asp Thr Ala
Gly Gln 5'  TGG GAY ACN GCN GGN CAR 3' 
Y C or T R G or A N G, A, T or C
Substitute Inosine for N
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Polymerase Chain Reaction
Asymmetric PCR different molar ratio of
primers for ssDNA amplification RT PCR Colony
PCR In situ PCR