Transcript No Slide Title

Restriction Enzyme
Digestion
Store in -20 C
Restriction Enzyme
Buffer
NaCl or KCl, TrisHCl, MgCl2, DTT
Different salt: varied activity
Buffer supplied with enzyme for 100 % activity
Restriction Enzyme
Buffer
Universal buffer for double or triple digestion
some activity reduced
Check manual from company
if different buffer to be used
Restriction Enzyme
Methylation
Modification commonly found in DNA
of bacteria, eukaryote and their virus
m6A, m5C, m4C, hm5C
Restriction Enzyme
Temperature
In general 37 C
67, 25 or 50 C also found
Restriction Enzyme
Enzyme inactivation
Heating 65 C for 15 min
Extraction with phenol/chloroform
Addition of EDTA
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 %
Electrophoresis
Analytical method for
purification / isolation
separation / fractionation
identification
Electrical field
Simple and Rapid
Gel medium / Buffer
Electrophoresis
Agarose / Polyacrylamide gel
Detection by staining
Ethidium bromide (UV)
Acridine orange (UV: ss-orange / ds-green)
Silver staining
Brilliant blue or Methylene blue
Range of Separation
Linear dsDNA Acrylamide Linear dsDNA
% in 1x TBE
%T in 1xTBE
(kb)
(bp)
0.3
5 - 60
3.5
100 - 1000
0.6
1 - 20
5.0
75 - 500
0.7
0.8 - 10
8.0
50 - 400
0.9
0.5 - 7
12.0
35 - 250
1.2
0.4 - 6
15.0
20 - 150
1.5
0.2 - 3
20.0
5 - 100
2.0
0.1 - 2
Agarose
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
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
Agarose Gel Electrophoresis
Agarose Gel Electrophoresis
Polyacrylamide Gel Electrophoresis
For protein analysis
For smaller DNA fragments
High resolving power
Run in vertical configuration
Polyacrylamide Gel Electrophoresis
Acrylamide and Bis-acrylamide
APS and TEMED without oxygen
Denaturing PAG with Urea/Formamide
for ssDNA
PAGE
PAGE
PAGE
Polymerase Chain Reaction
Temperature
Time
Denaturation
Annealing
Extension
Polymerase Chain Reaction
Components
Thermostable DNA polymerase
Template
Primer
Substrate
Buffer
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
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
Polymerase Chain Reaction
Polymerase Chain Reaction
Polymerase Chain Reaction
Thermal Cycler
Polymerase Chain Reaction
Hot start PCR
enzyme activity blocked during
reaction setup
enhance specificity / sensitivity
increase target yield
by wax, chemical or enzyme Ab
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
Polymerase Chain Reaction
Multiplex PCR
combined primer sets
amplify more than 1 target in 1 tube
Polymerase Chain Reaction
Real time PCR
quantify amplified products
comparative assay of initial templates
monitor increased fluorescence signal
during cycles (not end point)
Polymerase Chain Reaction
Touch-down / Step-down PCR
annealing temp progressively reduced
from above Tm to below Tm
specific target amplified at high stringency
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
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
Polymerase Chain Reaction
Asymmetric PCR
different molar ratio of primers
for ssDNA amplification
RT PCR
Colony PCR
In situ PCR