Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye...

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• Put 2 µl loading dye in n tubes • Transfer 8 µl of each reaction to a tube containing loading dye • Load on 1% gel and run at 150 volts using H3/R1 digest and 2 log ladder as markers • Use outcome to decide whether to clone or retry on Wed

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Transcript of Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye...

Page 1: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

• Put 2 µl loading dye in n tubes• Transfer 8 µl of each reaction to a tube containing

loading dye • Load on 1% gel and run at 150 volts using H3/R1

digest and 2 log ladder as markers• Use outcome to decide whether to clone or retry on Wed

Page 2: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Sequencing technologies Gene Regulation•Ion Torrent Trancriptional repressors•Illumina Circular RNA•Pyrosequencing (454) Long non-coding RNA•Solid RNA transcriptional activators•Pacific Bio miRNA•Nanopore Pol II pausing

Pol IV and Pol VChromatin remodeling

Digital (Droplet) PCR RNA localizationRNA degradationRNA terminationProtein degradationMetabolomicsMito/Cp gene regulation

http://www.biotechniques.com/news/

Page 3: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

MetabolomicsIdentifying all the metabolites in a given tissue• GC/MS for non-polars• LC/MS for polarsAltered levels of metabolites are often earliest clues to diseasehttp://www.bbc.co.uk/news/science-environment-22013700http://www.plosone.org/article/info%3Adoi

%2F10.1371%2Fjournal.pone.0059909

http://www.bbc.co.uk/news/science-environment-22013700
Page 4: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

How to make a cell?Must put all the right pieces in all the right places

Page 5: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

How to make a cell?Must put all the right pieces in all the right placesSome mt & cp proteins contain subunits encoded by organelle’s genome

Page 6: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Cytoplasmic inheritance1)first seen as strictly maternally inherited albino variegation• no linkage to nuclear genes• albinism strictly determined by the mother

Page 7: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Cytoplasmic inheritance1)first seen as strictly maternally inherited albino variegation• no linkage to nuclear genes• albinism strictly determined by the mothervariegation arises because have mix of “good”

and “bad” cp• Segregate randomly at division• eventually one form predominates

Page 8: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Cytoplasmic inheritanceVariegation arises because have mix of “good” and “bad” cp•Segregate randomly at division•eventually one form predominates

In plants, cytoplasm comes from the egg•most pollen do not have cp or mt•can't study genetically, because no way to mix parental organelles

Page 9: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAvary between 120 & 217 kB, according to species• most are 120-160 kB• have >20 copies/chloroplast• encode ~ 100 proteins, 4 rRNA &~30 tRNA

Page 10: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAencode ~ 100 proteins, 4 rRNA &~30 tRNA5 classes of proteins

1. ribosomal & other proteins involved in translation

2. proteins involved in transcription3. proteins involved in photosynthesis4. proteins involved in respiration• ORFs (open reading frames)sequences capable ofencoding proteins but no product has been identified

Page 11: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAencode ~ 100 proteins, 4 rRNA &~30 tRNA5 classes of proteins

in general, tend to be the more hydrophobic subunitscould have complicated exporting the gene to the nucleus

invariably also have subunits encoded by nuclear genes

Page 12: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAcpDNA encodes rubisco large subunit, nDNA encodes small subunit, holoenzyme has 8 lg & 8 small subunits

Page 13: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAcp gene expression is regulated at all levels

1) transcriptional 2) mRNA stability3) Translational: light triggers 100x increase in

some proteins but only small increase in transcription

Page 14: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAcoordination with nucleus•primarily studied during light-regulated cp development•light triggers development of proplastids •assemble thylakoids, make nearly all the proteins needed for photosynthesis

Page 15: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAcoordination with nucleus•primarily studied during light-regulated cp development•nucleus controls by sending in proteins including DNA polymerases and proteases•cp degrade excess subunits

Page 16: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAcoordination with nucleus•cp degrade excess subunits•when poison rbcS, rbcL is made but does not accumulate•same when poison rbcL with chloramphenicol

Page 17: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAcoordination with nucleusCP signals to nucleus: retrograde signaling•ROS•Redox•Mg-protoporphyrin•Genome-uncoupled (gun) mutants are defective in retrogradesignaling

Page 18: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Plastid DNAOddities

1) many cp genes have intronsintrons are self-splicing (type II): no spliceosomes or other enzymes!

2) mRNA editing:many cp mRNAs differ from the gene encoding them•an ACG is modified post-transcriptionally to a functional AUG start codon in several tobacco mRNAs; many other post-transcriptional changes have also been identified•editing machinery is encoded by the nucleus

Page 19: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mito DNA range from 200 to 2500 kb (cf 16 kb for mammalian mito)•7 fold variation in mt genome size within cucurbit family•watermelon =330 kb, muskmelon = 2500 kb•considerable variation within same species•5 different cytotopes in maize, vary from 540-700kb

Page 20: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mito DNA range from 200 to 2500 kb (cf 16 kb for mammalian mito)• reason for large size is unknown• encodes ~ 35 proteins, also rRNA & tRNA

• subunits of ATP synthase & complexes I, II, III & IV

Page 21: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mito DNA encodes ~ 35 proteins, also rRNA & tRNA

• subunits of ATP synthase & complexes I, II, III & IV• some mRNA are trans-spliced from 2 diff transcripts!

Page 22: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mito DNA encodes ~ 35 proteins, also rRNA & tRNA

• subunits of ATP synthase & complexes I, II, III & IV• some mRNA are trans-spliced from 2 diff transcripts!• some mRNA are edited: bases changed after synthesis!

Page 23: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mito DNA encodes ~ 35 proteins, also rRNA & tRNA

• subunits of ATP synthase & complexes I, II, III & IV• some mRNA are trans-spliced from 2 diff transcripts!• some mRNA are edited: bases changed after synthesis!•Mech to prevent nucleus from stealing genes?

•Find cp & nuc genes in mtDNA!

Page 24: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mitochondrial DNA• some mRNA are trans-spliced from 2 diff transcripts!• some mRNA are edited: bases changed after synthesis!•Mech to prevent nucleus from stealing genes?• mtDNA recombines to form new genes: see many smaller molecules cf one big circle

Page 25: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mitochondrial DNA•mtDNA recombines to form new genes: see many smaller molecules cf one big circle: some poison pollen development to create cytoplasmic male sterility

Page 26: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mitochondrial DNA• mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility•Pollen don't transmit mito!

Page 27: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mitochondrial DNA• mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility•Pollen don't transmit mito!•May be due to PCD (apoptosis)

Page 28: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mitochondrial DNA• mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility•Pollen don't transmit mito!•May be due to PCD (apoptosis)•Only have seenendoG in plant mt

Page 29: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

Mitochondrial DNA• mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility•Pollen don't transmit mito!•Widely used in plant breeding

•Eg hybrid corn

Page 30: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

CMS• mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility•described in over 150 different spp.can affect either sporophytic or gametophytic tissueeither pollen or tapetum can blow up

Page 31: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

CMS• mtDNA recombines to form new genes, some poison pollen development to create cytoplasmic male sterility•described in over 150 different spp.can affect either sporophytic or gametophytic tissueeither pollen or tapetum can blow uphave major increase in respiration and# mitochondria after meiosis40 x increase in mt/ cell in tapetum20x in sporogenous cells

Page 32: Put 2 µl loading dye in n tubes Transfer 8 µl of each reaction to a tube containing loading dye Load on 1% gel and run at 150 volts using H3/R1 digest.

CMSeither pollen or tapetum can blow uphave major increase in respiration and# mitochondria after meiosis40 x increase in mt/ cell in tapetum20x in sporogenous cellscan (usually) be overcome by nuclear "restorer" genes usually a single dominant gene


In vivo sledování doxorubicinu ve svalové tkáníweb2.mendelu.cz/af_239_nanotech/data/phd/2.seminar.pdf · Chicken muscle tissue ... Max intensity 50 µl 400 µl 100 µl 150 µl

In vivo sledování doxorubicinu ve svalové tkáníweb2.mendelu.cz/af_239_nanotech/data/phd/2.seminar.pdf · Chicken muscle tissue ... Max intensity 50 µl 400 µl 100 µl 150 µl

Practical 3. Analyzing DNA fragments · the components of your digests on ice ... 6 12 µl 2 µl 2 µl 2 µl 1 µl ... Flick the tube with your finger to mix well and spin for 5 sec

Practical 3. Analyzing DNA fragments · the components of your digests on ice ... 6 12 µl 2 µl 2 µl 2 µl 1 µl ... Flick the tube with your finger to mix well and spin for 5 sec

1.Set up 110 µl mix for each primer/DNA combo on ice! 1.1.1 µl 100x F primer (1 pMol/µl = 1µM final []) 2.1.1 µl 100x R primer 3.11 µl 10x PCR buffer 4.2.2.

1.Set up 110 µl mix for each primer/DNA combo on ice! 1.1.1 µl 100x F primer (1 pMol/µl = 1µM final []) 2.1.1 µl 100x R primer 3.11 µl 10x PCR buffer 4.2.2.

AriaMx Real-Time PCR Software...Primer (200-600 nM) X µL X µL Probe (150-600 nM) None X µL Template X µL X µL DW Up to 20 µL Up to 20 µL AriaMxリアルタイムPCRシステムの反応液量は10

AriaMx Real-Time PCR Software...Primer (200-600 nM) X µL X µL Probe (150-600 nM) None X µL Template X µL X µL DW Up to 20 µL Up to 20 µL AriaMxリアルタイムPCRシステムの反応液量は10

CONTENTS - Pipette 73002X Grey 1-ch 0.2–10 µl 0.05 µl 10 µl N/A N/A 73004X Yellow 1-ch 5–120 µl 0.50 µl 300 µl, 350 µl 721008 721018 73006X Orange 1-ch 10–300 µl 1.00

CONTENTS - Pipette 73002X Grey 1-ch 0.2–10 µl 0.05 µl 10 µl N/A N/A 73004X Yellow 1-ch 5–120 µl 0.50 µl 300 µl, 350 µl 721008 721018 73006X Orange 1-ch 10–300 µl 1.00

PRECISION LIQUID HANDLING - Microlab Tech · 2010-06-22 · MICROLAB ® Robotic ... 55019-09 100 µL 200 µL 21 and 22 55019-11 200 µL 200 µL 21 and 22 55019-13 250 µL 300 µL

PRECISION LIQUID HANDLING - Microlab Tech · 2010-06-22 · MICROLAB ® Robotic ... 55019-09 100 µL 200 µL 21 and 22 55019-11 200 µL 200 µL 21 and 22 55019-13 250 µL 300 µL

Agilent 6470 Triple Quadrupole LC/MS System and EMR-Lipid ...EMR vs QuEChERS workflow 400 µL extract + 600 µL water in autosampler vial. (Spinach: 200 µL extract + 800 µL water)

Agilent 6470 Triple Quadrupole LC/MS System and EMR-Lipid ...EMR vs QuEChERS workflow 400 µL extract + 600 µL water in autosampler vial. (Spinach: 200 µL extract + 800 µL water)

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Minicircle DNA Technology · 1 µl 10x T4 DNA ligase buffer 6 µl deionized water 1 µl T4 DNA ligase (40 U/ µl) 10 µl Total Volume b. Incubate the ligation reaction at 16°C overnight.

Minicircle DNA Technology · 1 µl 10x T4 DNA ligase buffer 6 µl deionized water 1 µl T4 DNA ligase (40 U/ µl) 10 µl Total Volume b. Incubate the ligation reaction at 16°C overnight.

MultiPurpose Sampler MPS MPS robotic MPS liquid · MultiPurpose Sampler MPS MPS robotic MPS roboticpro MPS liquid. 2 ... 50 µL 100 µL 250 µL 500 µL ... 500 l 1100111-805-00

MultiPurpose Sampler MPS MPS robotic MPS liquid · MultiPurpose Sampler MPS MPS robotic MPS roboticpro MPS liquid. 2 ... 50 µL 100 µL 250 µL 500 µL ... 500 l 1100111-805-00

Primerdesign Ltd TM Mycoplasma · 2019-01-07 · 2 x 105 per µl 2 x 104 per µl 2 x 103 per µl 2 x 102 per µl 20 per µl 2 per µl Standard Curve Copy Number Tube 1 Positive control

Primerdesign Ltd TM Mycoplasma · 2019-01-07 · 2 x 105 per µl 2 x 104 per µl 2 x 103 per µl 2 x 102 per µl 20 per µl 2 per µl Standard Curve Copy Number Tube 1 Positive control

SofTouch...8707-06XX 8-Channel 5 - 100 µl1 µl 200 µl, 300 µl 8707-07XX 8-Channel 25 - 250 µl5 µl 300 µl 8707-08XX 8-Channel 50 - 1200 µl 10 µl 1200 µl 8707-09XX 12-Channel

SofTouch...8707-06XX 8-Channel 5 - 100 µl1 µl 200 µl, 300 µl 8707-07XX 8-Channel 25 - 250 µl5 µl 300 µl 8707-08XX 8-Channel 50 - 1200 µl 10 µl 1200 µl 8707-09XX 12-Channel

Research Article Effect of Solvent, Dye-Loading Time, and ...Research Article Effect of Solvent, Dye-Loading Time, and Dye Choice on the Performance of Dye-Sensitized Solar Cells FahdM.Rajab

Research Article Effect of Solvent, Dye-Loading Time, and ...Research Article Effect of Solvent, Dye-Loading Time, and Dye Choice on the Performance of Dye-Sensitized Solar Cells FahdM.Rajab

Transfer 5 µl from your PCR tube to fresh tube, add 1 µl dye & run on 0.7% gel

Transfer 5 µl from your PCR tube to fresh tube, add 1 µl dye & run on 0.7% gel

VIDAS® ASSAYS - PRACTICAL INFORMATION…شخصات... · AFP 100 µL E2II 200 µL AMH 200 µL PRL 200 µL LH 200 µL FSH 200 µL PRG 200 µL CEAS 200 µL 199 200 µL 125 200 µL

VIDAS® ASSAYS - PRACTICAL INFORMATION…شخصات... · AFP 100 µL E2II 200 µL AMH 200 µL PRL 200 µL LH 200 µL FSH 200 µL PRG 200 µL CEAS 200 µL 199 200 µL 125 200 µL

pET Express & Purify Kits User Manual - Takara Bio Manual/PT5018-1.pdf15 µl pET6xHN-C Vector (In-Fusion Ready) [100 ng/µl] 10 µl pET6xHN-GFPuv Vector [500 ng/µl] 15 µl 1.1 kb

pET Express & Purify Kits User Manual - Takara Bio Manual/PT5018-1.pdf15 µl pET6xHN-C Vector (In-Fusion Ready) [100 ng/µl] 10 µl pET6xHN-GFPuv Vector [500 ng/µl] 15 µl 1.1 kb

Fine Care Biosystems - ENDO...VARIABLE 0.1 - 2.5 µl 0.5 - 10 µl 2 - 20 µl FIXED 2.5, 5, 10 µl MUL TI 0.5 - 10 µl VARIABLE 5 - 50 µl FIXED 20, 25 µl MULTI 5 - 50 µl VARIABLE

Fine Care Biosystems - ENDO...VARIABLE 0.1 - 2.5 µl 0.5 - 10 µl 2 - 20 µl FIXED 2.5, 5, 10 µl MUL TI 0.5 - 10 µl VARIABLE 5 - 50 µl FIXED 20, 25 µl MULTI 5 - 50 µl VARIABLE

ExpresswayŽ Linear Expression - Thermo Fisher Scientific · 2.5X IVPS Plus E. coli Reaction Buffer 20 µl T7 Enzyme Mix 1 µl 75 mM Methionine 1 µl Total Volume 42 µl Note: ...

ExpresswayŽ Linear Expression - Thermo Fisher Scientific · 2.5X IVPS Plus E. coli Reaction Buffer 20 µl T7 Enzyme Mix 1 µl 75 mM Methionine 1 µl Total Volume 42 µl Note: ...

c a c O ROSIE CHEEKS simon dye MELON simon dye JELLYBEAN ... · SPRING RAIN simon dye CLOUDY SKY simon dye SUNKIST simon dye CATKIN simon dye SCUBA simon dye WISTERIA simon dye O.c

c a c O ROSIE CHEEKS simon dye MELON simon dye JELLYBEAN ... · SPRING RAIN simon dye CLOUDY SKY simon dye SUNKIST simon dye CATKIN simon dye SCUBA simon dye WISTERIA simon dye O.c

20202017 ALEXMK2 Quick Guide A4...ALEX2 Stop Solution 8 Min 100 µL 400 µL 100 µL 500 µL 500 µL 500 µL 500 µL 500 µL Title 20202017_ALEXMK2_Quick_Guide_A4 Created Date 2/17/2020

20202017 ALEXMK2 Quick Guide A4...ALEX2 Stop Solution 8 Min 100 µL 400 µL 100 µL 500 µL 500 µL 500 µL 500 µL 500 µL Title 20202017_ALEXMK2_Quick_Guide_A4 Created Date 2/17/2020