lecture_notes:04-14-2010
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lecture_notes:04-14-2010 [2010/04/15 05:40] jstjohn created |
lecture_notes:04-14-2010 [2010/04/19 18:25] (current) galt |
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| + | ===== Library Construction ====== | ||
| {{:lecture_notes:235_library_construction.pdf|Nader's Powerpoint}} (Converted to PDF) | {{:lecture_notes:235_library_construction.pdf|Nader's Powerpoint}} (Converted to PDF) | ||
| + | ==== General Overview ==== | ||
| + | |||
| + | Current library generation methods more or less follow this general set of steps. | ||
| + | |||
| + | - Double stranded sample either cut up randomly or by some targeted approach. | ||
| + | - The overhanging ends are either finished with polymerase (if on the right strand) or digested with exonuclease. | ||
| + | - The blunt ended fragments are A tailed specifically on the 3' ends of the DNA. | ||
| + | - Double stranded library adapters have overhanging T which allows ligation to blunt ends in a specific orientation. | ||
| + | - This orientation may be used to ensure single stranded samples | ||
| + | |||
| + | ==== 454 Specific Library Construction ==== | ||
| + | |||
| + | ==== SOLiD Specific Library Construction ==== | ||
| + | Since you are dealing with a short sequence you need a larger quantity of material to start due to loss in the gell size selection phase. | ||
| + | |||
| + | === Mate Pair Library === | ||
| + | |||
| + | ==== Illumina Specific LIbrary Construction ==== | ||
| + | |||
| + | === Paired End Library === | ||
| + | |||
| + | ==== Targeted RNA Amplification ==== | ||
| + | |||
| + | Capable of amplifying with high specificity with low (~2%) ribosomal RNA contamination. | ||
| + | |||
| + | - First Strand cDNA synthesis | ||
| + | - Second strand cDNA synthesis | ||
| + | - SPIA Amplification | ||
| + | - RNAse H cuts up the RNA (SPIA) adapter because it is bound to the now DNA poly-A tail of the original RNA sample, and strips away some of one of the strands of cDNA randomly. | ||
| + | - Anneal back on an RNA SPIA primer (with attached RNA sequence to re-bind to the DNA poly-A tail for starting over from the first part of this step) | ||
| + | - Extend the cDNA sequence back out to its fullest extent with polymerase, adding the remainder of the partially degraded cDNA strand to the linearly growing pool of cDNA sample. | ||
| + | - Add RNAse H and continue with these steps until desired quantity is achieved. | ||
| + | |||
lecture_notes/04-14-2010.1271310037.txt.gz ยท Last modified: 2010/04/15 05:40 by jstjohn
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