Data Received – Geoduck Genome Sequencing by Illumina

We previously sent some geoduck samples to Illumina, as part of a pilot project for them to test out a new sequencing platform. The data has finally arrived!

It was sent on a 4TB Seagate external hard drive.

Due to weird connection issues we’ve recently encountered with our server, Owl (Synology DS1812+), I connected the HDD directly to Owl via USB (instead of connecting to a computer and transferring). I transferred the data using the Synology web interface to avoid any computer/NAS connection issues that might interrupt the transfer.

We have a meeting with the Illumina people tomorrow afternoon to review the data they’ve provided (looks like it’s going to take awhile, though). Once that meeting takes place, we’ll figure out how to document this project in our data management plan.

 

 

 

Synthesis of 3-bromo-5-chloro-[1,2,4]triazolo[4,3-a]pyrazine

Reaction Scheme:

Step3ReactionScheme.png

Compound

MW (g/mol)

Mol (mmol)

Mass (g)

Density (g/mL)

Volume (mL)

Equiv

5-chloro-[1,2,4]triazolo[4,3-a]pyrazine

154

1.69

0.26

 

 

1

NBS

177.98

1.52

0.281

 

 

0.9

Chloroform

 

 

 

 

15

 

3-bromo-5-chloro-[1,2,4]triazolo[4,3-a]pyrazine

231.92

1.69

0.392

 

 

1

 

Procedure:

6/14/17 NBS was recrystallized before the reaction. Water (35 ml) was added to NBS (3 g). The mixture was stirred and heated at 100 celsius degree until NBS was dissolved to make a yellow solution. The solution was cooled to room temperature. White crystals formed. The crystals were filtered out and washed with ice water. The crystals were placed on the high vac in a dessicator over P2O5 to dry overnight.

7/5/17 5-chloro-[1,2,4]triazolo[4,3-a]pyrazine (0.26 g, 2 mmol) and N-bromosuccinimide (0.281 g, 1.6 mmol, 0.9equiv.) were placed in chloroform (50 mL) and refluxed for 44 h. Reflux started at 10:30 am.

7/6/17 TLC showed that there was still starting material. Another 0.5 equivalents of NBS were added to the reaction. TLC at 2:00 pm still showed a lot of starting material.

7/7/17 TLC still showed mostly starting material. There seems to be a faint product spot. Another 0.5 equivalents of NBS were added, but TLC at the end of the day was still the same, so reaction was stopped. Flask was kept covered with aluminum foil to block light.

BrClfinalTLC.png

7/19/17 Solvent removed under reduced pressure. NMR showed that some product had formed, but there was still unreacted starting material.

NMRcrudeBrCl.png

7/21/17 Product was purified by column chromatography using 70:30 EtOAc: Hexanes. The NBS came off first, followed by the product. Solvent was removed under reduced pressure to yield a yellow solid (0.37 g, 1.6 mmol, 94% yield). NMR obtained.

BrClFinalNMR.png

Synthesis of 5-chloro-[1,2,4]triazolo[4,3-a]pyrazine (JSLT2-2)

Reaction Scheme:

Screen Shot 2017-07-27 at 11.39.36 AM.png

Compound 

MW (g/mol) 

Mol (mmol) 

Mass (g) 

Density (g/mL) 

Volume (mL) 

Equiv

2-chloro-6-hydrazinylpyrazine 

144.56 

3.14 

0.454 

 

 

1 

Toluene  

 

 

 

 

10 

 

Triethyl Orthoformate 

148.2 

6.28 

0.931 

0.891 

1.04 

2.0 

TsOH 

190.22 

0.157 

0.03 

 

 

0.05 

Product 

154 

 

 

 

 

1 

Procedure:

6/28/17 Hydrazinylpyrazine (0.454 g, 3.14 mmol, 1 equiv.) triethyl orthoformate (0.931 g 1.04 ml, 6.28 mmol, 2 equiv.), and tosylic acid (0.0299 mg, 0.157  mmol, 0.05 equiv.) were dissolved in toluene (10 ml) and heated at  reflux for about 24 hours 

TsOH placed on high vac in a dessicator under P2Oovernight. Reaction started at 3:30. Hydrazinylpyrazine was dissolved in toluene first. It dissolved around 65 degrees. Triethyl Orthoformate and TsOH were then added. The temperature was turned up to 95 degrees, and reaction was left overnight. TLC was taken in 30:70 EtOAc: Hexanes at 5:00 pm and indicated that some starting material remained. 

6/29/17 Reaction removed from reflux at 10:30 am. TLC in 50:20 EtOAc: Hexanes showed starting material was gone and one of the intermediates had disappeared. We think that the spot just below the starting material is probably the product. Reaction mixture turned very dark brown. Solvent removed under reduced pressure to yield a reddish brown solid (0.523 g, 3.40 mmol, 108% yield). 

6/30/17 Column ran with 70:30 EtOAc: Hexanes as the starting eluent. Gradient ran up to 100% EtOAc. The bottom spot on our TLC, which we assumed to be the product was fluorescent blue on the bottom half of the spot and dark brown on the top. The blue and brown eluted separately from the column (blue followed by brown).  

Fractions 27-59 (brown spot) and Fractions 61-80 were collected. Solvents was removed under reduced pressure. NMR of both fractions were obtained.  

NMR indicated that the product was in fractions 27-59 (0.31 g, 2.0 mmol, 64 % yield) 

TLC: 

6/28/17 5:00pm

Solvent System( on left):TLC in 30:70 EtOAc: Hexanes

Solvent system( on right): 50:20  EtOAc: Hexanes

Screen Shot 2017-07-27 at 11.47.25 AM.png

NMR:

Screen Shot 2017-07-27 at 11.51.26 AM.png

Screen Shot 2017-07-27 at 11.51.35 AM.png

Synthesis of 2-Chloro-6-hydrazinylpyrazine (JSLT 1-2)

Reaction Scheme: 

Reaction Scheme.png

Compound 

MW (g/mol) 

Mol (mmol) 

Mass (g) 

Density (g/mL) 

Volume (mL) 

Equiv 

2,6-Dichloropyrazine 

148.98 

13.4 

2 

 

 

1 

Hydrazine hydrate  

50.06 

13.4 

0.680 

1.02 

0.66 

1 

EtOH 

 

 

 

 

20 

 

Product: 2-chloro-6-hydrazinylpyrazine 

144.56 

13.4 

 

 

 

1 

Procedure: 

6/19/17 2,6-Dichloropyrazine (2 g, 13.4 mmol) was stirred in ethanol (20 mL) and hydrazine hydrate (0.66 mL, 680 mg, 13.4 mmol) was added. The mixture was heated at 80˚C for 12 h. 

Reflux started at 3 pm.  

6/20/17 Reaction was removed from the heat at 9:30 am. TLC showed a small amount of starting material, but most of it was gone.  

Br:ClS1TLC.png

The solvent was removed under reduced pressure to yield a yellow solid. The solid was dissolved in 30mL water and 40 mL ethyl acetate. The aqueous layer was extracted with 3x20mL etoac. The organic layers were combined and washed with 20 mL brine, and then dried over MgSO4. Solvent was removed under reduced pressure to yield yellow solid (1.05 g, 7.26 mmol, 54% yield)

S1GCMS.png


The first step to synthesis of 2-chloro-6-hydrazinylpyrazine(1 and redo 1 )

Synthesis of 2-chloro-6-hydrazinylpyrazine #1synthesis of blah from blah

Procedure:

 

Compound

Amount

(mg or mL)

n (mmol)

eq.

MW

density

 (if liquid)

2,6-dichloropyrazine

7.45g

50

1

 149.00g/mol

 

EtOH

100ml

 

 

 46.07g/mol

 

hydrazine

9.3oml

25

2

32.05g/mol 

 

EtOAc

150ml

 

 

88.11 g/mol 

 

water

75ml

 

 

18.02 g/mol 

 

2-chloro-6-hydrazineylpyrazine

g

 

 

 

 

2,6-dichloropyrazine(7.45g) was dissolved in EtOH (100 mL, 0.50 M 3 %), and hydrazine (9.30 mL, 2 eq) added. The reaction mixture was stirred at reflux for 1 hours.

 

The solvent was removed under reduced pressure. Water (75 mL) and EtOAc (150mL) were added, and the mixture was shaken in an attempt to dissolve all solid. The organic layer was removed and the aqueous layer was washed 3 times with EtOAc.

 

An insoluble brown solid was filtered off and aqueous layer was washed 3 times with EtOAc.

The product is about ?g. It is yellow crystal and it is ?%. It may have the impurity( the possible structure

impurity.png
).

 

Risk assesment

Risk Assesment YANG.pdf

 

DATA

 Because of some reasons , this lab do not have any TLC's picture 

NMR: 

step1 BY.pdf
     
Document 1analysis.mnova

 

Compound strings

<?xml version="1.0" encoding="GBK"?><cml xmlns="http://www.chemaxon.com" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.chemaxon.com/marvin/schema/mrvSchema_16_02_15.xsd" version="ChemAxon file format v16.02.15, generated by v17.14.0">
<MHead><MarvinGUI><mprop name="saveproperties" dataType="xsd:boolean" value="true"></mprop><mprop name="aminoAcidBondColoringEnabled" dataType="xsd:boolean" value="true"></mprop><mprop name="automaticReactionEnabled" dataType="xsd:boolean" value="true"></mprop><mprop name="setColoringEnabled" dataType="xsd:boolean" value="true"></mprop><mprop name="atomNumberingType" dataType="xsd:integer" value="0"></mprop><mprop name="fogFactor" dataType="xsd:integer" value="66"></mprop><mprop name="automaticFogEnabled" dataType="xsd:boolean" value="false"></mprop><mprop name="atomPropertiesVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="atomMappingVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="EZVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="mpVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="bondLength" dataType="xsd:double" value="28.0"></mprop><mprop name="bondLengthVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="valenceErrorVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="valenceErrorVisibleInView" dataType="xsd:boolean" value="false"></mprop><mprop name="atomSymbolVisible3D" dataType="xsd:boolean" value="true"></mprop><mprop name="rgroupsVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="lonePairsAutoCalc" dataType="xsd:boolean" value="true"></mprop><mprop name="lonePairsAsLine" dataType="xsd:boolean" value="false"></mprop><mprop name="chargeWithCircle" dataType="xsd:boolean" value="false"></mprop><mprop name="molbg" value="#ffffff"></mprop><mprop name="chiralitySupport" dataType="xsd:integer" value="0"></mprop><mprop name="atomsize" dataType="xsd:double" value="0.35714285714"></mprop><mprop name="boldBondWidth" dataType="xsd:double" value="5.0"></mprop><mprop name="bondSpacing" dataType="xsd:double" value="0.180375"></mprop><mprop name="bondHashSpacing" dataType="xsd:double" value="0.15"></mprop><mprop name="wireThickness" dataType="xsd:double" value="0.066666"></mprop><mprop name="stickThickness" dataType="xsd:double" value="0.1"></mprop><mprop name="ballRadius" dataType="xsd:double" value="0.5"></mprop><mprop name="rendering" value="wireframe"></mprop><mprop name="downWedge" value="mdl"></mprop><mprop name="coordinateBondStyle" value="arrow"></mprop><mprop name="coordinateBondStyleAtMulticenter" value="hashed"></mprop><mprop name="anybond" value="auto"></mprop><mprop name="sketchAnyBond" value="auto"></mprop><mprop name="viewAnyBond" value="auto"></mprop><mprop name="atomFont" dataType="xsd:mfont" value="SansSerif-PLAIN-10"></mprop><mprop name="sketchImplicitH" value="heteroterm"></mprop><mprop name="lonePairsVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="autoscale" dataType="xsd:boolean" value="false"></mprop><mprop name="absLabelVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="colorScheme" value="cpk"></mprop><mprop name="viewCarbonVisibility" value="inChain"></mprop><mprop name="sketchCarbonVisibility" value="inChain"></mprop><mprop name="viewLigandOrderVisibility" value="showOnlyWithDefinition"></mprop><mprop name="sketchLigandOrderVisibility" value="showOnlyWithDefinition"></mprop><mprop name="rLogicVisible" value="off"></mprop><mprop name="peptideDisplayType" value="3-letter"></mprop><mprop name="zeroBasedAtomIndexing" dataType="xsd:boolean" value="false"></mprop><mprop name="valencePropertyVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="ligandErrorVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="sketchArrowHeadLength" dataType="xsd:double" value="0.8"></mprop><mprop name="sketchArrowHeadWidth" dataType="xsd:double" value="0.5"></mprop><mprop name="sketchArrowTailLength" dataType="xsd:double" value="0.8"></mprop><mprop name="sketchArrowTailWidth" dataType="xsd:double" value="0.5"></mprop><mprop name="scale" dataType="xsd:double" value="18.0"></mprop></MarvinGUI></MHead>
<MDocument><MChemicalStruct><molecule molID="m1"><atomArray atomID="a1 a2 a3 a4 a5 a6 a7 a8" elementType="Cl Cl C C N C C N" x2="-15.2493126776983 -20.584019384069276 -16.582996689835532 -16.582996689835532 -17.91666603088379 -19.250335371932046 -19.250335371932046 -17.91666603088379" y2="3.206675097163168 3.206675097163168 2.4366835674892773 0.8966496863711964 0.1266327458121559 0.8966496863711964 2.4366835674892773 3.2067005080483177"></atomArray><bondArray><bond id="b1" atomRefs2="a3 a1" order="1"></bond><bond id="b2" atomRefs2="a4 a3" order="1"></bond><bond id="b3" atomRefs2="a5 a4" order="2"></bond><bond id="b4" atomRefs2="a6 a5" order="1"></bond><bond id="b5" atomRefs2="a7 a2" order="1"></bond><bond id="b6" atomRefs2="a7 a6" order="2"></bond><bond id="b7" atomRefs2="a8 a3" order="2"></bond><bond id="b8" atomRefs2="a8 a7" order="1"></bond></bondArray></molecule></MChemicalStruct></MDocument>
</cml>

to

<?xml version="1.0" encoding="GBK"?><cml xmlns="http://www.chemaxon.com" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.chemaxon.com/marvin/schema/mrvSchema_16_02_15.xsd" version="ChemAxon file format v16.02.15, generated by v17.14.0">
<MHead><MarvinGUI><mprop name="saveproperties" dataType="xsd:boolean" value="true"></mprop><mprop name="aminoAcidBondColoringEnabled" dataType="xsd:boolean" value="true"></mprop><mprop name="automaticReactionEnabled" dataType="xsd:boolean" value="true"></mprop><mprop name="setColoringEnabled" dataType="xsd:boolean" value="true"></mprop><mprop name="atomNumberingType" dataType="xsd:integer" value="0"></mprop><mprop name="fogFactor" dataType="xsd:integer" value="66"></mprop><mprop name="automaticFogEnabled" dataType="xsd:boolean" value="false"></mprop><mprop name="atomPropertiesVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="atomMappingVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="EZVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="mpVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="bondLength" dataType="xsd:double" value="28.0"></mprop><mprop name="bondLengthVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="valenceErrorVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="valenceErrorVisibleInView" dataType="xsd:boolean" value="false"></mprop><mprop name="atomSymbolVisible3D" dataType="xsd:boolean" value="true"></mprop><mprop name="rgroupsVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="lonePairsAutoCalc" dataType="xsd:boolean" value="true"></mprop><mprop name="lonePairsAsLine" dataType="xsd:boolean" value="false"></mprop><mprop name="chargeWithCircle" dataType="xsd:boolean" value="false"></mprop><mprop name="molbg" value="#ffffff"></mprop><mprop name="chiralitySupport" dataType="xsd:integer" value="0"></mprop><mprop name="atomsize" dataType="xsd:double" value="0.35714285714"></mprop><mprop name="boldBondWidth" dataType="xsd:double" value="5.0"></mprop><mprop name="bondSpacing" dataType="xsd:double" value="0.180375"></mprop><mprop name="bondHashSpacing" dataType="xsd:double" value="0.15"></mprop><mprop name="wireThickness" dataType="xsd:double" value="0.066666"></mprop><mprop name="stickThickness" dataType="xsd:double" value="0.1"></mprop><mprop name="ballRadius" dataType="xsd:double" value="0.5"></mprop><mprop name="rendering" value="wireframe"></mprop><mprop name="downWedge" value="mdl"></mprop><mprop name="coordinateBondStyle" value="arrow"></mprop><mprop name="coordinateBondStyleAtMulticenter" value="hashed"></mprop><mprop name="anybond" value="auto"></mprop><mprop name="sketchAnyBond" value="auto"></mprop><mprop name="viewAnyBond" value="auto"></mprop><mprop name="atomFont" dataType="xsd:mfont" value="SansSerif-PLAIN-10"></mprop><mprop name="sketchImplicitH" value="heteroterm"></mprop><mprop name="lonePairsVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="autoscale" dataType="xsd:boolean" value="false"></mprop><mprop name="absLabelVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="colorScheme" value="cpk"></mprop><mprop name="viewCarbonVisibility" value="inChain"></mprop><mprop name="sketchCarbonVisibility" value="inChain"></mprop><mprop name="viewLigandOrderVisibility" value="showOnlyWithDefinition"></mprop><mprop name="sketchLigandOrderVisibility" value="showOnlyWithDefinition"></mprop><mprop name="rLogicVisible" value="off"></mprop><mprop name="peptideDisplayType" value="3-letter"></mprop><mprop name="zeroBasedAtomIndexing" dataType="xsd:boolean" value="false"></mprop><mprop name="valencePropertyVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="ligandErrorVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="sketchArrowHeadLength" dataType="xsd:double" value="0.8"></mprop><mprop name="sketchArrowHeadWidth" dataType="xsd:double" value="0.5"></mprop><mprop name="sketchArrowTailLength" dataType="xsd:double" value="0.8"></mprop><mprop name="sketchArrowTailWidth" dataType="xsd:double" value="0.5"></mprop><mprop name="scale" dataType="xsd:double" value="18.0"></mprop></MarvinGUI></MHead>
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</cml>

 

ClC1=CN=CC(Cl)=N1

to

NNC1=NC(Cl)=CN=C1



The first step to synthesis of 2-chloro-6-hydrazinylpyrazine

Synthesis of 2-chloro-6-hydrazinylpyrazinesynthesis of blah from blah

Procedure:

 

2,6-dichloropyrazine(2.1g) was dissolved in EtOH (28 mL, 0.50 M 3 %), and hydrazine (1.35 mL, 2 eq) added. The reaction mixture was stirred at reflux for 16 hours.

 

The solvent was removed under reduced pressure. Water (20 mL) and EtOAc (~55mL) were added, and the mixture was shaken in an attempt to dissolve all solid. The organic layer was removed and the aqueous layer was washed 3 times with EtOAc.

 

An insoluble brown solid was filtered off and aqueous layer was washed 3 times with EtOAc.

Risk assesment

Risk Assesment YANG.pdf

 

DATA

 

Because of some reasons , this lab do not have any TLC's picture 

NMR: 

email_DK-1_1_1.pdf

 

Compound strings

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to

<?xml version="1.0" encoding="GBK"?><cml xmlns="http://www.chemaxon.com" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.chemaxon.com/marvin/schema/mrvSchema_16_02_15.xsd" version="ChemAxon file format v16.02.15, generated by v17.14.0">
<MHead><MarvinGUI><mprop name="saveproperties" dataType="xsd:boolean" value="true"></mprop><mprop name="aminoAcidBondColoringEnabled" dataType="xsd:boolean" value="true"></mprop><mprop name="automaticReactionEnabled" dataType="xsd:boolean" value="true"></mprop><mprop name="setColoringEnabled" dataType="xsd:boolean" value="true"></mprop><mprop name="atomNumberingType" dataType="xsd:integer" value="0"></mprop><mprop name="fogFactor" dataType="xsd:integer" value="66"></mprop><mprop name="automaticFogEnabled" dataType="xsd:boolean" value="false"></mprop><mprop name="atomPropertiesVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="atomMappingVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="EZVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="mpVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="bondLength" dataType="xsd:double" value="28.0"></mprop><mprop name="bondLengthVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="valenceErrorVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="valenceErrorVisibleInView" dataType="xsd:boolean" value="false"></mprop><mprop name="atomSymbolVisible3D" dataType="xsd:boolean" value="true"></mprop><mprop name="rgroupsVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="lonePairsAutoCalc" dataType="xsd:boolean" value="true"></mprop><mprop name="lonePairsAsLine" dataType="xsd:boolean" value="false"></mprop><mprop name="chargeWithCircle" dataType="xsd:boolean" value="false"></mprop><mprop name="molbg" value="#ffffff"></mprop><mprop name="chiralitySupport" dataType="xsd:integer" value="0"></mprop><mprop name="atomsize" dataType="xsd:double" value="0.35714285714"></mprop><mprop name="boldBondWidth" dataType="xsd:double" value="5.0"></mprop><mprop name="bondSpacing" dataType="xsd:double" value="0.180375"></mprop><mprop name="bondHashSpacing" dataType="xsd:double" value="0.15"></mprop><mprop name="wireThickness" dataType="xsd:double" value="0.066666"></mprop><mprop name="stickThickness" dataType="xsd:double" value="0.1"></mprop><mprop name="ballRadius" dataType="xsd:double" value="0.5"></mprop><mprop name="rendering" value="wireframe"></mprop><mprop name="downWedge" value="mdl"></mprop><mprop name="coordinateBondStyle" value="arrow"></mprop><mprop name="coordinateBondStyleAtMulticenter" value="hashed"></mprop><mprop name="anybond" value="auto"></mprop><mprop name="sketchAnyBond" value="auto"></mprop><mprop name="viewAnyBond" value="auto"></mprop><mprop name="atomFont" dataType="xsd:mfont" value="SansSerif-PLAIN-10"></mprop><mprop name="sketchImplicitH" value="heteroterm"></mprop><mprop name="lonePairsVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="autoscale" dataType="xsd:boolean" value="false"></mprop><mprop name="absLabelVisible" dataType="xsd:boolean" value="false"></mprop><mprop name="colorScheme" value="cpk"></mprop><mprop name="viewCarbonVisibility" value="inChain"></mprop><mprop name="sketchCarbonVisibility" value="inChain"></mprop><mprop name="viewLigandOrderVisibility" value="showOnlyWithDefinition"></mprop><mprop name="sketchLigandOrderVisibility" value="showOnlyWithDefinition"></mprop><mprop name="rLogicVisible" value="off"></mprop><mprop name="peptideDisplayType" value="3-letter"></mprop><mprop name="zeroBasedAtomIndexing" dataType="xsd:boolean" value="false"></mprop><mprop name="valencePropertyVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="ligandErrorVisible" dataType="xsd:boolean" value="true"></mprop><mprop name="sketchArrowHeadLength" dataType="xsd:double" value="0.8"></mprop><mprop name="sketchArrowHeadWidth" dataType="xsd:double" value="0.5"></mprop><mprop name="sketchArrowTailLength" dataType="xsd:double" value="0.8"></mprop><mprop name="sketchArrowTailWidth" dataType="xsd:double" value="0.5"></mprop><mprop name="scale" dataType="xsd:double" value="18.0"></mprop></MarvinGUI></MHead>
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ClC1=CN=CC(Cl)=N1

to

NNC1=NC(Cl)=CN=C1



RNA Isolation – Olympia oyster gonad tissue in paraffin histology blocks

My previous go at this was a little premature – I didn’t wait for Laura to fully annotate her slides/blocks. Little did I know, the tissue was mostly visceral mass and, as such, I didn’t hit much in the way of actual gonad tissue. So, I’m redoing this, now that Grace has gone through and annotated the blocks to point out gonad tissue. SN-10-16 was sent to Katherine Silliman on 20170720.

Isolated RNA from Olympia oyster gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. See Laura’s notebook for full details on samples and preservation.

 

RNA was isolated from the following samples using the PAXgene Tissue RNA Kit (Qiagen). Gouged samples from the blocks weighing ~10mg from each of the tissues and processed according the protocol for isolating RNA from blocks of paraffin-embedded tissues.

Background on all of this is in this GitHub Issue

NF-10-22
NF-10-23
NF-10-24
NF-10-26
NF-10-28
NF-10-30
SN-10-16
SN-10-17
SN-10-20
SN-10-25
SN-10-26
SN-10-31

IMPORTANT:

  • Prior to beginning, I prepared an aliquot of Buffer TR1 by adding 40μL of β-mercaptoethanol (β-ME) to 4000μL of Buffer TR1)

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:

  • “Max speed” spins were performed at 20,000g.
  • Tissue disruption was performed by adding ~25-50 glass beads (425 – 600μm diameter) with the Disruptor Genie @ 45C for 15mins (in the Friedman Lab).
  • Samples were eluted with 27μL of Buffer TR4 x 2, incubated @ 65C for 5mins, immediately placed on ice.

 

Results:

Samples were not quantified due to lack of proper RNA Qubit assay AND the computer that our NanoDrop1000 is hooked up to is dead. Will have Katherine Silliman perform quantification.

Samples were stored at -80C temporarily.

Samples will be sent to Katherine Silliman for high-throughput library construction and sequencing once I hear back from her regarding her availability to receive the samples.

Synthesis of 5-(4-chlorophenyl)-6-methylpyrimidine-2,4-diamine

Scheme:

AcetylScheme.png

SM/P 

(Z)-2-(4-chlorophenyl)-3-methoxybut-2-enenitrile 

Guanidine chloride 

Sodium methoxide 

5-(4-chlorophenyl)-6-methylpyrimidine-2-diamine 

 

mw 

207.66 

95.53 

54.02 

234.69 
 

Equiv 

1.00 

3.00 

3.00 

1.00 

 

mmol 

2.72 

8.16 

8.16 

2.72 

 

g 

0.565 

0.78 

0.44 

0.6 
 

mL 

 
 

 

 
 

 

g/mL 

 

 

1.059 

 

Procedure:

7/17/17 Guanidine hydrochloride (0.78 g, 8.16 mmol, 3 eq) and sodium methoxide (0.44 g, 8.16 mmol, eq) were dissolved in ethanol (5.8 mL) and stirred for 5 min. A solution of (Z)-2-(4-chlorophenyl)-3-methoxy-3-phenylacrylonitrile (0.565 g, 2.72 mmol, 1 eq) in ethanol (1.9 mL) was added to the cloudy, white solution. The reaction mixture was heated under reflux for 6 hours. Some starting material was lost from syringe during transfer.  

AcetylTLC.jpg

30:70 EtOAc: Hexanes, silica gel plate. Starting material gone after 6 hours of reflux.

7/18/17 The reaction mixture was diluted with dichloromethane. Saturated NaCl was added and the mixture was extracted with dichloromethane and then washed with saturated NaCl. Organic extracts were dried over MgSO4. Solvent was removed under reduced pressure to yield a white/yellow solid (0.157 g,0.67 mmol26 % yield).  

Product was crystallized from methanol and placed in the freezer overnight to yield white crystals (0.0845 g, 0.36 mmol, 14% yield).  

JS_LTDaraprimAcetylS3Crystals_PROTON_cdcl3_01.jpeg