1 / 66

Purifying DNA & RNA Source Amounts & Purity Damage or Loss

Purifying DNA & RNA Source Amounts & Purity Damage or Loss. Cells. Extract. Remove junk. Pure DNA. Bind DNA. 1. Remove high MW junk. DNA, RNA Solution. Denatured Protein. Phenol. Cell Extract. Shake. Spin. 2. Remove low MW junk and concentrate. Add. (to 70%). and salt.

ellery
Download Presentation

Purifying DNA & RNA Source Amounts & Purity Damage or Loss

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Purifying DNA & RNA Source Amounts & Purity Damage or Loss

  2. Cells Extract Remove junk Pure DNA Bind DNA

  3. 1. Remove high MW junk DNA, RNA Solution Denatured Protein Phenol Cell Extract Shake Spin

  4. 2. Remove low MW junk and concentrate Add (to 70%) and salt SPIN (fast)

  5. Or Bind DNA BIND + SALT (aided by alcohol) ELUTE - NO SALT (just add water)

  6. EXTRACT IN High Salt High Salt Wash WATER SILICA PARTICLES DNA

  7. Purifying one type of DNA away from other DNA molecules • Plasmids from bacterial chromosomal DNA • (Form) • phage DNA • (Location) • Restriction fragments, PCR products • (Size)

  8. SDS, alkali

  9. alkali neutralize

  10. RNA PURIFICATION Lyse & denature proteins FAST Acidic phenol Or bind glass/silica Small RNAs?

  11. A? B? C? M

  12. Oligo-dT beads for polyA+ mRNA

  13. High Salt

  14. Chemical Synthesis of oligonucleotides Uses? Block and unblock sequentially so that only one nucleotide adds at a time

  15. Phosphoramidite Protected amino groups * 5’ 3’ *

  16. Couple Blocked 5’-OH Growing chain

  17. Unblock 1st “nucleotide”

  18. Add and activate next “nucleotide”

  19. Couple

  20. Product 99%

  21. Cap 1%

  22. 1st nuc on bead. (blocked at 5’-OH) 3’- 5’ unblock Add next nuc. (blocked 5’) Remove from bead couple De-protect Unblock 5’-OH cap Purify

  23. RNA harder DNA variants like PNA

  24. If DNA is too large for conventional electrophoresis….

  25. Pulsed-field electrophoresis

  26. Polyacrylamide Gels can resolve small DNAs differing in length by one nucleotide

  27. Dideoxy sequencing converts sequence Information (A, C, G, T) into size differences e.g. if a DNA has T residues at positions 2, 5, 13, 16… this can be converted into a set of DNAs of length n+ 2, 5, 13, 16.. (which can be measured by denaturing polyacrylamide gel electrophoresis)

  28. ddCTP

  29. dATP ddATP dCTP dGTP dTTP LABEL

  30. 2,5,13,16 ddA ddC 9,10,15,19 ddG 1,4,7,8,12,14,20 ddT 3,6,11,17,18

  31. n + ddA 2,5,13,16 ddC 9,10,15,19 ddG 1,4,7,8,12,14,20 ddT 3,6,11,17,18

  32. 2,5,13,16 ddA ddC 9,10,15,19 ddG 1,4,7,8,12,14,20 ddT 3,6,11,17,18

  33. ddA ddC ddG ddT

  34. What do you need to sequence DNA? Where do the reagents come from? Must the DNA be pure? How much is needed How much good sequence can you obtain?

More Related