Restriction Enzyme Digestion Store in 20 C Restriction
- Slides: 35
Restriction Enzyme Digestion Store in -20 C
Restriction Enzyme Buffer Na. Cl or KCl, Tris. HCl, Mg. Cl 2, 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 m 6 A, m 5 C, m 4 C, hm 5 C
Restriction Enzyme Temperature In general 37 C 25 , 67 or 50 C also found
Restriction 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 p. H Substitution of Co 2+, Mn 2+, Zn 2+ for Mg 2+ 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 ds. DNA Acrylamide Linear ds. DNA % in 1 x TBE %T in 1 x. TBE (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 ss. DNA
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 u. M
Polymerase Chain Reaction d. NTPs 50 -200 u. M each recommended Mg. Cl 2 1. 5 m. M 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 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 ss. DNA amplification RT PCR Colony PCR In situ PCR
- Mechanism of restriction enzyme
- Cla 1 restriction enzyme
- Restriction enzyme
- Mechanism of restriction enzyme
- Restriction enzyme mapping
- Restriction enzymes
- Restriction enzyme mapping
- Restriction enzyme
- Restriction enzyme analysis of dna ap bio lab
- Illustration of the steps in restriction digestion and pcr
- What is restriction digestion
- Beta limit dextrin
- Venn diagram mechanical and chemical digestion
- Row store vs column store
- Row store vs column store
- Rflp analysis
- Symmetrical growth restriction
- Circular permutation with repetition
- Dna fingerprinting rflp
- Dna fragment length
- Symmetrical and asymmetrical iugr
- Function of restriction endonuclease
- Restriction enzymes
- Restriction mapping
- Pedigree vocabulary
- Extension activity 1 plasmid mapping answers
- Iugr causes
- Selective breeding definition biology
- Palindromic sequence
- Restriction fragment length polymorphism
- Iugr cause
- Molecular scissors wikipedia
- Heavy connotations
- Diffusion restriction
- Endonuclease restriction
- Texas transportation code 521.025