SLIDE 1
mRNA purification through fragmentation Distinct 28S ribosomal - - PowerPoint PPT Presentation
mRNA purification through fragmentation Distinct 28S ribosomal - - PowerPoint PPT Presentation
mRNA purification through fragmentation Distinct 28S ribosomal Distinct 18S subunit (or prok. 23S): ribosomal 28S ideally 2X size of 18S subunit (or prok. 16S) 18S No well defined peaks between Flat baseline Marker throughout
SLIDE 2
SLIDE 3
Distinct 28S ribosomal subunit (or prok. 23S): ideally 2X size of 18S Distinct 18S ribosomal subunit (or
- prok. 16S)
No well defined peaks between ribosomal peaks
Flat baseline throughout electropherogram
Marker
Small peaks are sometimes present after the marker at 24 – 29
- seconds. These are represented by 5S and 5.8S subunits, tRNAs, and
small RNA fragments about 100bp. These are especially noted when using phenol and trizol extraction methods. They can be removed by treating total RNA through Qiagen columns which removes small RNAs.
Marker 28S 18S ~100 bp
SLIDE 4
The 28S subunit often degrades first Decrease in ribosomal peak intensities Increase in intensity of smaller digested RNA fragments Baseline between and to the left
- f ribosomal peaks becomes jagged
Total RNA with images like this are borderline. Re-extraction should be seriously considered.
SLIDE 5
Combination of 5S, 5.8S, tRNAs, and an increase in digested RNAs Decrease in ribosomal peak intensities marker Digested RNA ~100 bp
SLIDE 6
~100 bp
SLIDE 7
Relies on base pairing between
poly-A tail and oligo dT beads
SLIDE 8
SLIDE 9
Fragmentation Buffer: Mg2+ and Heat Stop Solution: EDTA
SLIDE 10
Used for concentrating and de-salting
DNA or RNA in an aqueous solution
Components:
- Salt (NaAcO):
RNA has a negatively charge backbone NaAcO breaks up to Na+ and [CH3COO]- Salt ions neutralize the charge on the nucleic acids, the molecules become less hydrophillic and less soluable
- EtOH
Easier for Na+ to interact with the phosphate backbone of the nucleitides in EtOH than in water because EtOH is less polar
Image from: http://openwetware.org/wiki/Image:DNA_EtOH_precipitation_small_pellet.jpg
SLIDE 11
1st Strand Synthesis 2nd Strand Synthesis End Repair 3’ Adenylation Ligation Gel Extraction PCR Amplification
Same as DN DNA Proto tocol
SLIDE 12