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--- bdev:nucleic_acid_extraction [2020/11/29 19:07]
richard created
+++ bdev:nucleic_acid_extraction [2021/08/16 22:22] (current)
@@ Line 29: / Line 29: @@
 https://patents.google.com/patent/US20120107799A1/en?q=disposable+pipette+tip+extraction+nucleic+acid&scholar&oq=disposable+pipette+tip+extraction+nucleic+acid
+----
+Based on the review of some papers on this topic, we managed to put together a simple test to assess if it is possible to use Whatman paper to do a nucleic acid extraction out from a cell lysate.
+For this simple study two differents of cells were used. 1) Yeast cells and 2) E coli. The protocol from Lui et al recipes for replication (slightly modified):
+Lysis buffer - 80mM Tris Acetate, 2M guanidine hydrochloride, 100mM NaCl, 4mM EDTA, 1% SDS
+Wash buffer - 10mM Tris (pH 8.0), 0.1% SDS)
+Elution - Molecular grade H2O
+----
+Protocol
+  - 0.4cm^2 whatman paper cut and placed into sterile 1.5mL Eppendorf tubes (sandwiched standing perpendicular to the ground toward the bottom of the tube)
+  - 500uL Lysis buffered added to tubes
+  - 100ul O/N E.coli BL21 in LB broth added to tubes
+  - Tubes mixed by inversion with some light pipetting - for 1 - 1.5 minutes
+  - All Liquid removed by pipette or dumped over a Kimwipe
+  - 400uL Wash buffer added, mixed by inversion and light pipette -  for 1-1.5 minutes
+  - Tube dumped over Kimwipe and allowed to dry 2 minutes
+  - The Whatman paper was then used in the pcr reaction in a new .2mL PCR tube
+  - 150ul PCR grade H2O was washed over the membrane with a p1000 pipette for 30 seconds
+  - 2ul of the H2O supernatant was used for PCR amplification
+{{ :bdev:image-20200406-150430.png?200 |}}
+The yeast samples didn’t work because we believe it is more difficult to lyse these cells.
+One thing to note that you need to remove the Whatman,  paper from the solution in order to run the reaction. But this could be achieved in the pipette tip, it could be positioned further up the pipette and not exposed do the liquid after elution during the amplification/detection reaction.
+While it is true that you can amplify DNA directly from Ecoli culture/lysate too but the PCR reaction is not as efficient. This is shown here where the water elution is in lane 2 and lane 7 is a positive control, lane 8 is the negative control.
+Protocol for lanes:
+  - Ladder
+  - According to the protocol from gel #1
+  - Same protocol as lane 2, but with vortexing instead of mix by inversion
+  - Same protocol as lane 2 with and extra 75% ETOH wash before H2O elution
+  - Same protocol as lane 4 with vortexing instead of mixing by inversion
+  - Same protocol as lane 2, but removing the Whatman paper after the lysis step
+  - Pos Cntrl with kit extracted e.coli DNA
+  - Master mix neg cntrl
+{{ :bdev:22.jpg?200 |}}
+**All reaction prepared with same master mix of 515F and 1492R primers on E.coli O/N in LB media

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