Difference between revisions of "Team:Munich/Cas13a/test"
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Since microfluidic mixing of liquids is a rather complex process, we settled for an Isothermal PCR-based approach (RPA). | Since microfluidic mixing of liquids is a rather complex process, we settled for an Isothermal PCR-based approach (RPA). | ||
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<img style="width:30%" src="https://static.igem.org/mediawiki/2017/e/e0/T--Munich--pic--lysis_pcr_deltacells.png"> | <img style="width:30%" src="https://static.igem.org/mediawiki/2017/e/e0/T--Munich--pic--lysis_pcr_deltacells.png"> | ||
[1] log2 DNA ladder, [2] 10<sup>6</sup> cells/ml, [3] 10<sup>5</sup> cells/ml, [4] 5*10<sup>4</sup> cells/ml, [5] 10<sup>4</sup> cells/ml,[6] 5*10<sup>3</sup> cells/ml, [7] 10<sup>3</sup> cells/ml, [8] 10<sup>2</sup> cells/ml,[9] 10<sup>6</sup> cells/ml (no heat-lysis step. only PCR at 37 °C) | [1] log2 DNA ladder, [2] 10<sup>6</sup> cells/ml, [3] 10<sup>5</sup> cells/ml, [4] 5*10<sup>4</sup> cells/ml, [5] 10<sup>4</sup> cells/ml,[6] 5*10<sup>3</sup> cells/ml, [7] 10<sup>3</sup> cells/ml, [8] 10<sup>2</sup> cells/ml,[9] 10<sup>6</sup> cells/ml (no heat-lysis step. only PCR at 37 °C) | ||
Revision as of 18:05, 1 November 2017
Cell lysis and RNA extraction
For RNA extraction from our bacterial targets, we looked at several possible lysis methods. We tried and abandoned Guanidine-salts as lysis agent, since its strong chaotropic power makes extensive purification necessary. For the same reason regarding the need for purification, although to a lesser extent, we used detergent/heat lysis only in our lab work. While we investigated RNA-silica binding properties (see labbook Sept. 1st to 5th, section "other") and tested commercial silica-based kits for such purifications, we decided against adding unnecessary complexity for our prototype.
Alkaline lysis is well-known for DNA-, but not for RNA-extraction due to rapid degradation thereof under alkaline conditions. Since our protein responds to a very short part of our target sequence (<30 bp), compared to the resulting RNA fragments (most >300 pb), it should work none the less and with better efficiency and superior speed (seconds) compared to detergent/heat lysis.
Since microfluidic mixing of liquids is a rather complex process, we settled for an Isothermal PCR-based approach (RPA). With the exceptional sensitivity of PCR, we can even use an inefficient heat-only lysis (5 to 10 times less efficient than Detergent/Heat for e. coli in our comparisons) and still detect RNA with an amount of 100 cells in the PCR reaction volume (PCR band measurable from column 4: 5*104 cells/ml * 2 mikroL = 100 cells).
[1] log2 DNA ladder, [2] 106 cells/ml, [3] 105 cells/ml, [4] 5*104 cells/ml, [5] 104 cells/ml,[6] 5*103 cells/ml, [7] 103 cells/ml, [8] 102 cells/ml,[9] 106 cells/ml (no heat-lysis step. only PCR at 37 °C)
