DNA Extraction – Ava Withering Syndrome Transmission Study Tissues

Isolated DNA from 117 tissue samples that I weighed out yesterday.

DNA was extracted using the QIAmp Fast DNA Stool Mini Kit (Qiagen) following the manufacturer’s protocol with the following options:

  • Samples were briefly homogenized (due to their stiffness resulting from ethanol fixation) in the InhibitEX Buffer using disposable plastic pestles.
  • Homogenized tissue was incubated at 95C to maximize cell lysis
  • Followed “human DNA analysis” protocol for remainder of protocol (to maximize sample recovery)
  • Eluted DNA with 100μL Buffer ATE

Samples were stored at 4C in FSH240 in racks for qPCR and quantification next week.

Sample information is in this spreadsheet (Google Sheet): 20170502_Ava_Ab_List

Synthesis of OSM-S-106

Attempts at synthesizing OSM-S-106:

http://malaria.ourexperiment.org/aminotpseries/5827/Suzuki_coupling_using_the_same_conditions_that_are_used_to_generate_boronate_esters_JRC_502.html >28% yield

http://malaria.ourexperiment.org/aminotpseries/10415/Synthesis_of_34aminothieno32dpyrimidin6ylbenzenesulfonamide_PT23_AKA_OSMS106.html 90.5% yield

http://malaria.ourexperiment.org/aminotpseries/10468/Synthesis_of_34aminothieno32dpyrimidin6ylbenzenesulfonamide_AEW_1721_OSMS106.html 



Synthesis of OSM-S-154

Attempts at synthesizing OSM-S-154:

http://malaria.ourexperiment.org/aminotpseries/5713/Synthesis_of_3sulfamoylphenylboronic_acid_JRC_294.html 31% yield

http://malaria.ourexperiment.org/aminotpseries/4677/Synthesis_of_3sulfamoylphenylboronic_acid_JRC_491_META_ISOMER.html 61% yield

http://malaria.ourexperiment.org/aminotpseries/4666/Synthesis_of_4sulfamoylphenylboronic_acid_JRC_491_PARA_ISOMER.html 56% yield

http://malaria.ourexperiment.org/aminotpseries/4665/Synthesis_of__2sulfamoylphenylboronic_acid_JRC_481_ORTHO_ISOMER.html 22% yield

http://malaria.ourexperiment.org/aminotpseries/4038/Upscaled_synthesis_of_34455tetramethyl132dioxaborolan_2ylbenzenesulfonamide_JRC_292.html 52% yield

http://malaria.ourexperiment.org/aminotpseries/4219/Upscaled_synthesis_of_3sulfamoylphenylboronic_acid_JRC_293.html 56% yield

http://malaria.ourexperiment.org/aminotpseries/7619/Synthesis_of_34455tetramethyl132dioxaborolan2ylbenzenesulfonamide_MJT_111.html did not work



Synthesis of OSM-S-139

Attempts at synthesizing OSM-S-139:

http://malaria.ourexperiment.org/aminotpseries/4284/Synthesis_of_6bromothieno32dpyrimidin4amine_JRC_401.html 43% yield

http://malaria.ourexperiment.org/aminotpseries/8963/Synthesis_of_6Bromothieno32dpyrimidin4amine_CT_34.html 43% yield

http://malaria.ourexperiment.org/aminotpseries/8887/Synthesis_of_6Bromothieno32dpyrimidin4amine_CT_33.html 42% yield

http://malaria.ourexperiment.org/aminotpseries/8663/Synthesis_of_6Bromothieno32dpyrimidin4amine_CT_32.html 45% yield

http://malaria.ourexperiment.org/aminotpseries/11450/Amination_of_Bromochlorothienopyrimidine_TF151.html 60.4% yield

http://malaria.ourexperiment.org/aminotpseries/6938/Amination_of_bromochlorothienopyrimidine_AT64.html 76% yield

http://malaria.ourexperiment.org/aminotpseries/6861/Amination_of_bromochlorothienopyrimidine_AT62.html 55% yield

http://malaria.ourexperiment.org/aminotpseries/6861/Amination_of_bromochlorothienopyrimidine_AT62.html 55% yield

http://malaria.ourexperiment.org/aminotpseries/6857/Amination_of_bromochlorothienopyrimidine_AT61.html 47% yield

http://malaria.ourexperiment.org/aminotpseries/6341/Synthesis_of_6bromothieno32dpyrimid4amine_JRC_402.html 78% yield

http://malaria.ourexperiment.org/aminotpseries/3113/Synthesis_of_thieno32dpyrimidin4amine_JRC_141.html 63% yield

http://malaria.ourexperiment.org/aminotpseries/3169/Synthesis_of_thieno32dpyrimidin4amine_JRC_142.html 94% yield

http://malaria.ourexperiment.org/aminotpseries/3187/Synthesis_of_thieno32dpyrimidin4amine_JRC_143.html 

http://malaria.ourexperiment.org/aminotpseries/3197/Synthesis_of_thieno32dpyrimidin4amine_JRC_144.html 

http://malaria.ourexperiment.org/aminotpseries/8111/Synthesis_of_6Bromothieno32dpyrimidin4amine_AEW_1001.html did not work

http://malaria.ourexperiment.org/aminotpseries/8159/Synthesis_of_6Bromothieno32dpyrimidin4amine_AEW_1002.html did not work

http://malaria.ourexperiment.org/aminotpseries/8169/Synthesis_of_6Bromothieno32dpyrimidin4amine_AEW_1003.html did not work

http://malaria.ourexperiment.org/aminotpseries/8220/Synthesis_of_6Bromothieno32dpyrimidin4amine_AEW_1005.html 

http://malaria.ourexperiment.org/aminotpseries/8185/Synthesis_of_6Bromothieno32dpyrimidin4amine_AEW_1004.html 

http://malaria.ourexperiment.org/aminotpseries/10457/ynthesis_of_6Bromothieno32dpyrimidin4amine_AEW_1006.html 

http://malaria.ourexperiment.org/aminotpseries/10165/Amination_of_bromochlorothienopyrimidine_PT21.html 

http://malaria.ourexperiment.org/aminotpseries/9145/Synthesis_of_6Bromothieno32dpyrimidin4amine_CT_37.html 


Synthesis of OSM-S-121

Attempts at synthesizing OSM-S-121:

http://malaria.ourexperiment.org/aminotpseries/4372/JRC_273.html 53% yield

http://malaria.ourexperiment.org/aminotpseries/4254/Synthesis_of_4chloro6bromothieno32dpyrimidine_via_BuLi_272.html 50% yield

http://malaria.ourexperiment.org/aminotpseries/7040/Bromination_of_chlorothienopyrimidine_AT56.html 64% yield

http://malaria.ourexperiment.org/aminotpseries/10148/Synthesis_of_6Bromo4chlorothieno32dpyrimidine_PT19.html 64.8% yield

http://malaria.ourexperiment.org/aminotpseries/6863/Bromination_of_chlorothienopyrimidine_AT53.html 39% yield

http://malaria.ourexperiment.org/aminotpseries/6852/Bromination_of_chlorothienopyrimidine_AT52.html 32% yield

http://malaria.ourexperiment.org/aminotpseries/6820/Bromination_of_chlorothienopyrimidine_AT51.html 55% yield

http://malaria.ourexperiment.org/aminotpseries/6314/Synthesis_of_4chloro6bromothieno32dpyrimidine_via_BuLi_AB31.html 70% yield

http://malaria.ourexperiment.org/aminotpseries/3670/Synthesis_of_6bromothieno32dpyrimidin4amine_212.html 15% yield

http://malaria.ourexperiment.org/aminotpseries/5859/Synthesis_of_6bromo4chlorothieno32dpyrimidine_JRC_275.html did not work

http://malaria.ourexperiment.org/aminotpseries/6536/Synthesis_of_4chloro6bromothieno32dpyrimidine_via_BuLi_AB32.html 12.3% yield

http://malaria.ourexperiment.org/aminotpseries/7039/Bromination_of_chlorothienopyrimidine_AT55.html 56% yield

http://malaria.ourexperiment.org/aminotpseries/8103/Synthesis_of_6Bromo4chlorothieno32dpyrimidine_AEW_991.html 56% yield


Synthesis of OSM-S-70

Attempts at synthesizing OSM-S-70: 

http://malaria.ourexperiment.org/aminotpseries/6079/Upscaled_synthesis_of_4chlorothieno32dpyrimidine_JRC_136.html  60% yield

http://malaria.ourexperiment.org/aminotpseries/5826/Upscaled_synthesis_of_4chlorothieno32dpyrimidine_JRC_135.html  48% yield

http://malaria.ourexperiment.org/aminotpseries/3040/Synthesis_of_4chlorothieno32dpyrimidine_JRC_131.html  29% yield

http://malaria.ourexperiment.org/aminotpseries/3194/Upscaled_synthesis_of_4chlorothieno32dpyrimidine_JRC_133.html  67% yield

http://malaria.ourexperiment.org/aminotpseries/6261/Synthesis_of_4chlorothieno32dpyrimidine_AB21.html 73% yield

http://malaria.ourexperiment.org/aminotpseries/3096/Synthesis_of_4chlorothieno32dpyrimidine_JRC_132.html 78% yield



Goals – May 2017

A day late, but definitely not a dollar short!

No goals posted last month because I didn’t want anyone to think they were just an April Fool’s joke. ;)

This month my goal is to continue my domination of Pub-a-Thon 2017!

IMG_0238_PNG

 

I plan on doing so by getting a second paper submitted this month! That’s right! I’m working on getting the following paper re-submitted:

Differential response to stress in Ostrea lurida (Carpenter 1864)

 

Synthesis of OSM-S-171

Attempt at synthesizing OSM-S-171:

http://malaria.ourexperiment.org/aminotpseries/6218/Synthesis_of_Thieno32dpyrimidine43Hone__AB11.html  67% yield

http://malaria.ourexperiment.org/aminotpseries/2660/Resynthesis_of_Thieno32dpyrimidine43Hone_JRC_82.html  17% yield

http://malaria.ourexperiment.org/aminotpseries/2535/Synthesis_of_Thieno32dpyrimidine43Hone_JRC_81.html did not work yield

http://malaria.ourexperiment.org/aminotpseries/2754/Synthesis_of_Thieno32dpyrimidine43Hone_using_formic_acid_JRC_91.html did not work yield

http://malaria.ourexperiment.org/aminotpseries/2976/Synthesis_of_Thieno32dpyrimidine43Hone_using_formic_acid_and_formamide_JRC_92.html 31% yield

http://malaria.ourexperiment.org/aminotpseries/2989/Upscaled_synthesis_of_Thieno32dpyrimidine43Hone_using_formic_acid_and_formamide_JRC_93.html 42% yield

http://malaria.ourexperiment.org/aminotpseries/3009/Upscaled_Synthesis_of_Thieno32dpyrimidine43Hone_using_formic_acid_and_formamide_JRC_94.html 25% yield



DNA Quantification – RLO viability DNased RNA

I previously DNased RNA I isolated from water filters that were part of the RLO viability experiment that Lisa and the Capstone students are conducting. I checked for residual gDNA carryover via qPCR and all of the samples that were intended for dosing the abalone came up positive. It’s likely due to such a high quantity of algae that was co-filtered with the potential RLOs, leading to over-saturation of the RNAzol with DNA, resulting in the gDNA carryover.

In turn, I think the DNase treatment was insufficient for the quantity of carryover DNA.

I am planning on re-DNasing those samples, but want to quantify any residual DNA present to make sure that the samples aren’t still too concentrated for the DNase.

Samples were quantified using the Robert Lab Qubit 3.0 and the Qubit dsHS reagents (high sensitivity), using 1uL of sample.

Results:

Residual DNA is still present, but at levels that are well below the maximum that the DNase treatment (10ug) can handle. I will redo the DNase treatment on these samples. Spreadsheet is linked, and embedded below, with sample concentrations.

Spreadsheet (Google Sheet): 20170424_filter_rna_dna_quant