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Achmad Farajallah | DNA Extraction Sri Bulan Musmiah G352110101Copyright Achmad Farajallah [email protected]://achamad.staff.ipb.ac.id/2011/11/28/dna-extraction-sri-bulan-musmiah-g352110101/
DNA Extraction Sri Bulan Musmiah G352110101
EKSTRAKSI DNASri Bulan Musmiah G352110101
NO SPECIES
SAMPLE DNA EXTRACTION GENOMETARGET
1 Moa(Aves:Dinornithiformes)
Bonepowder
DNA was extracted from 200 mgof bone powder by incubationwith rotation at 55°C for 48 h in1.5 mL digestion buffer [20 mMTris, pH 8.0, 1% Triton X-100,10mM dithiotheitol (DTT), 1mg/mL proteinase K and 0.47 MEDTA].The supernatant was spunthrough 30,000 MWCO Vivaspincolumns and then combined with5 volumes of PBI buffer (Qiagen,Valencia, CA) before the DNA wasextracted using silica spincolumns (Qiagen).
Mitochondrialcontrolregion Totalgenome
2 Cows,pronghorn,
bison ,black-tailed
prairiedogs
page 1 / 107
Dung Fresh dung (pellets or portions ofdung patties) was collected byobserving focal individuals untilthey defecated. Samples werecollected in a clean resealablebag. These bags were kept in aniced cooler and transported backto lab within 24 h, then stored at-20°C.Frozen dung samples werecut using a sterile scalpel, andapproximately 0.25 g of dung wasteased out from the middle ofseveral freshly cut subsamples.
Fungaltotalgenome
3 Birds Feather DNA extraction of thearchaeological feather followedthe same protocols as the freshsamples. The protocols followsstrict contamination-control forthe analysis of ancient remains.Two and five feather barbs wereremoved from the feather shaft.Barbs were first rinsed in 3%sodium hypochlorite for 30seconds to remove possiblesurface contamination, then
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Achmad Farajallah | DNA Extraction Sri Bulan Musmiah G352110101Copyright Achmad Farajallah [email protected]://achamad.staff.ipb.ac.id/2011/11/28/dna-extraction-sri-bulan-musmiah-g352110101/
(Fungi?)
DNA was extracted using theUltraClean® Fecal DNA isolationkit
rinsed twice in DNase/RNase-freedistilled water. Afterdecontamination, two differentDNA-extraction techniques wereapplied.The samples firstunderwent an MSSC extractionprotocol designed for degradedDNA samples. As keratinis themajor structural component offeather barbs, we modified thelysis buffer to includedithiothreitol (DTT). Barbs wereincubated in 1.5 to 3 ml of lysisbuffer (0.05 mol/l EDTA pH 8.0,0.5% SDS, 0.5 mg/ml proteinase Kand 10 mg/ml DTT) at 55°C for 1to 2 hours. The remainder of theDNA extraction producing a finalelution of 100 µl of DNA solutionfor each sample. Subsequently,the feather-barb subsamples werealso extracted using a commercialkit producing a final elution of 100µl of DNA solution for eachsample.
Fragments 200 to300 bp insize ofthemitochondrial DNAcytochrome bgene
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4 Cow Milkandcurd
Total DNA was isolated from milksamples by a phenol/chloroformmethod, followed by ethanolprecipitation according toSambrook et al.(1989)Cheesesamples were cut into smallpieces, and total DNA
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Achmad Farajallah | DNA Extraction Sri Bulan Musmiah G352110101Copyright Achmad Farajallah [email protected]://achamad.staff.ipb.ac.id/2011/11/28/dna-extraction-sri-bulan-musmiah-g352110101/
Totalgenome -fragmentedgenome
page 3 / 107
from cheese matrix was obtainedby performingphenol/chlorophorm as describedabove.
DNA from reference strain ofbacteria were prepared from pureculture according to theguanidium extraction proceduredescribed by Pitcher et al. (1989).
Totalgenome,includemicrobialgenom
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page 4 / 107
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5
page 5 / 107
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page 6 / 107
Clams
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Achmad Farajallah | DNA Extraction Sri Bulan Musmiah G352110101Copyright Achmad Farajallah [email protected]://achamad.staff.ipb.ac.id/2011/11/28/dna-extraction-sri-bulan-musmiah-g352110101/
Muscletissue
page 7 / 107
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Achmad Farajallah | DNA Extraction Sri Bulan Musmiah G352110101Copyright Achmad Farajallah [email protected]://achamad.staff.ipb.ac.id/2011/11/28/dna-extraction-sri-bulan-musmiah-g352110101/
Individuals with opened andclosed shells were preservedfromeach beach in 80% ethanol.Muscle tissue was extracted fromthe middle and apex region (c.1mm2) of the foot and cleanedwith ethanol (75%) to removesand, detritusor external organicmatter.DNA extraction wasperformed with the QiagenDNAMini kit, with slighltymodification to increase theconcentration of DNA, i.e. weused 150 µl of elution buffer.
page 8 / 107
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Achmad Farajallah | DNA Extraction Sri Bulan Musmiah G352110101Copyright Achmad Farajallah [email protected]://achamad.staff.ipb.ac.id/2011/11/28/dna-extraction-sri-bulan-musmiah-g352110101/
Fragmentof themitochondrial COIgene
totalgenome
page 9 / 107
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6
page 10 / 107
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page 11 / 107
Seacucumber
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Oraltentacle
page 12 / 107
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Upon collection, specimens werecleaned in seawater, immediatelyfrozen on dry ice (-70°C).Typically 200 mg of oral tentaclewas ground in liquid N2 in thepresence of 600 µl of proteinasesolution (50 mM Tris–HCl, pH 7.5,50 mM EDTA, pH 8.0, 0.4% SDS,0.5 mg/ml Proteinase K). Theground samples wereimmediately placed at 65°C forminimum of 2 h. The digestedsamples were repeteadlyextracted withphenol:chloroform:isoamylalcohol, 25:24:1. The resultantaqueous phase was adjusted to0.5 M NaCl and to 1%cetyltrimethylammonium bromide(CTAB) and incubated for a further20 min at 65°C. After two finalphenol/chloroform extractions,total DNA was precipitated byadding an equal volume ofisopropanol followed bysedimentation at 13,000 rpm for20 min. The DNA pellet was rinsedwith 500 µl of 70% ethanol,air-dried, and resuspended in 200µl of TE buffer (10 mM Tris–HCl,pH 7.5, 1 mM EDTA).
page 13 / 107
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16S
mitochondrialribosomalDNAfragments
totalgenome
page 14 / 107
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page 15 / 107
7
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Snake
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0C.Sloughed skins, mostly collectedat the entrance of identified adderdomains, were preserved dry atroom temperature for up to twoyears. An additional fresh sloughwas obtained from an adderobserved in the process ofecdysis. Snake faeces (associatedwith sloughs) collected from arange of UK sites were eitherimmediately frozen at -20oC orpreserved in 95% ethanol. A 10 µlblood sample, obtained by caudalextraction from an adder andstored in 90 µl of Seutin's buffer(Seutin et al.,1991) at roomtemperature, was collected as apositive PCR control.Sloughedskin required a rehydration stepto remove impurities prior to DNAextraction. A fragment (1-2 cm2)of slough was rinsed in 1 ml ofddwater at 55 oC in a rockingincubator for 4-6 h, and repeatedfor a further 8-12 h. DNAextraction was performed onthese rehydrated samples, eggyolks (approximately 0.2 cm3),NIS muscle (1 cm3) and blood (5µl in Seutin'sbuffer) usingQiagenDNeasy tissue extractionkit. Faecal material was extractedusing QIAamp@ DNA stool minikit.
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TM (YasuiKikai,Osaka, Japan). The nailpowder was lysed in a urea-lysissolution (2 M urea; 0.5 %SDS; 10mM Tris-HCl, pH 7.5; 0.1 M EDTA)containing 1 mg/ml proteinase Kand 40 mM DDT at 55 °Covernight. Nail DNA was extractedwith phenol/chloroform, andprecipitated with ethanol andsodium acetate. Precipitated nailDNA was dissolved again inextraction buffer (0.5 % SDS; 10mM Tris-HCl, pH 7.5; 0.1 M EDTA)containing 1 mg/ml proteinaseK, and incubated at 55 °Covernight. DNA was purifiedagainas above, and was suspended in30 µl of 1x TE buffer.
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EKSTRAKSI DNASri Bulan Musmiah G352110101
NO SPECIES
SAMPLE DNA EXTRACTION GENOMETARGET
1 Moa(Aves:Dinornithiformes)
Bonepowder
DNA was extracted from 200 mgof bone powder by incubationwith rotation at 55°C for 48 h in1.5 mL digestion buffer [20 mMTris, pH 8.0, 1% Triton X-100,10mM dithiotheitol (DTT), 1mg/mL proteinase K and 0.47 MEDTA].The supernatant was spunthrough 30,000 MWCO Vivaspincolumns and then combined with5 volumes of PBI buffer (Qiagen,Valencia, CA) before the DNA wasextracted using silica spincolumns (Qiagen).
Mitochondrialcontrolregion Totalgenome
2 Cows,pronghorn,
bison ,black-tailed
prairiedogs
Dung Fresh dung (pellets or portions ofdung patties) was collected byobserving focal individuals untilthey defecated. Samples werecollected in a clean resealablebag. These bags were kept in aniced cooler and transported backto lab within 24 h, then stored at-20°C.Frozen dung samples werecut using a sterile scalpel, andapproximately 0.25 g of dung wasteased out from the middle ofseveral freshly cut subsamples.
Fungaltotalgenome
3 Birds Feather DNA extraction of thearchaeological feather followedthe same protocols as the freshsamples. The protocols followsstrict contamination-control forthe analysis of ancient remains.Two and five feather barbs wereremoved from the feather shaft.Barbs were first rinsed in 3%sodium hypochlorite for 30seconds to remove possiblesurface contamination, then
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page 37 / 107
4 Cow Milkandcurd
Total DNA was isolated from milksamples by a phenol/chloroformmethod, followed by ethanolprecipitation according toSambrook et al.(1989)Cheesesamples were cut into smallpieces, and total DNA
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Totalgenome -fragmentedgenome
page 38 / 107
from cheese matrix was obtainedby performingphenol/chlorophorm as describedabove.
DNA from reference strain ofbacteria were prepared from pureculture according to theguanidium extraction proceduredescribed by Pitcher et al. (1989).
Totalgenome,includemicrobialgenom
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page 39 / 107
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5
page 40 / 107
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page 41 / 107
Clams
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Muscletissue
page 42 / 107
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Individuals with opened andclosed shells were preservedfromeach beach in 80% ethanol.Muscle tissue was extracted fromthe middle and apex region (c.1mm2) of the foot and cleanedwith ethanol (75%) to removesand, detritusor external organicmatter.DNA extraction wasperformed with the QiagenDNAMini kit, with slighltymodification to increase theconcentration of DNA, i.e. weused 150 µl of elution buffer.
page 43 / 107
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Fragmentof themitochondrial COIgene
totalgenome
page 44 / 107
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6
page 45 / 107
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page 46 / 107
Seacucumber
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Oraltentacle
page 47 / 107
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Upon collection, specimens werecleaned in seawater, immediatelyfrozen on dry ice (-70°C).Typically 200 mg of oral tentaclewas ground in liquid N2 in thepresence of 600 µl of proteinasesolution (50 mM Tris–HCl, pH 7.5,50 mM EDTA, pH 8.0, 0.4% SDS,0.5 mg/ml Proteinase K). Theground samples wereimmediately placed at 65°C forminimum of 2 h. The digestedsamples were repeteadlyextracted withphenol:chloroform:isoamylalcohol, 25:24:1. The resultantaqueous phase was adjusted to0.5 M NaCl and to 1%cetyltrimethylammonium bromide(CTAB) and incubated for a further20 min at 65°C. After two finalphenol/chloroform extractions,total DNA was precipitated byadding an equal volume ofisopropanol followed bysedimentation at 13,000 rpm for20 min. The DNA pellet was rinsedwith 500 µl of 70% ethanol,air-dried, and resuspended in 200µl of TE buffer (10 mM Tris–HCl,pH 7.5, 1 mM EDTA).
page 48 / 107
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16S
mitochondrialribosomalDNAfragments
totalgenome
page 49 / 107
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page 50 / 107
7
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Snake
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0C.Sloughed skins, mostly collectedat the entrance of identified adderdomains, were preserved dry atroom temperature for up to twoyears. An additional fresh sloughwas obtained from an adderobserved in the process ofecdysis. Snake faeces (associatedwith sloughs) collected from arange of UK sites were eitherimmediately frozen at -20oC orpreserved in 95% ethanol. A 10 µlblood sample, obtained by caudalextraction from an adder andstored in 90 µl of Seutin's buffer(Seutin et al.,1991) at roomtemperature, was collected as apositive PCR control.Sloughedskin required a rehydration stepto remove impurities prior to DNAextraction. A fragment (1-2 cm2)of slough was rinsed in 1 ml ofddwater at 55 oC in a rockingincubator for 4-6 h, and repeatedfor a further 8-12 h. DNAextraction was performed onthese rehydrated samples, eggyolks (approximately 0.2 cm3),NIS muscle (1 cm3) and blood (5µl in Seutin'sbuffer) usingQiagenDNeasy tissue extractionkit. Faecal material was extractedusing QIAamp@ DNA stool minikit.
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TM (YasuiKikai,Osaka, Japan). The nailpowder was lysed in a urea-lysissolution (2 M urea; 0.5 %SDS; 10mM Tris-HCl, pH 7.5; 0.1 M EDTA)containing 1 mg/ml proteinase Kand 40 mM DDT at 55 °Covernight. Nail DNA was extractedwith phenol/chloroform, andprecipitated with ethanol andsodium acetate. Precipitated nailDNA was dissolved again inextraction buffer (0.5 % SDS; 10mM Tris-HCl, pH 7.5; 0.1 M EDTA)containing 1 mg/ml proteinaseK, and incubated at 55 °Covernight. DNA was purifiedagainas above, and was suspended in30 µl of 1x TE buffer.
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EKSTRAKSI DNASri Bulan Musmiah G352110101
NO SPECIES
SAMPLE DNA EXTRACTION GENOMETARGET
1 Moa(Aves:Dinornithiformes)
Bonepowder
DNA was extracted from 200 mgof bone powder by incubationwith rotation at 55°C for 48 h in1.5 mL digestion buffer [20 mMTris, pH 8.0, 1% Triton X-100,10mM dithiotheitol (DTT), 1mg/mL proteinase K and 0.47 MEDTA].The supernatant was spunthrough 30,000 MWCO Vivaspincolumns and then combined with5 volumes of PBI buffer (Qiagen,Valencia, CA) before the DNA wasextracted using silica spincolumns (Qiagen).
Mitochondrialcontrolregion Totalgenome
2 Cows,pronghorn,
bison ,black-tailed
prairiedogs
Dung Fresh dung (pellets or portions ofdung patties) was collected byobserving focal individuals untilthey defecated. Samples werecollected in a clean resealablebag. These bags were kept in aniced cooler and transported backto lab within 24 h, then stored at-20°C.Frozen dung samples werecut using a sterile scalpel, andapproximately 0.25 g of dung wasteased out from the middle ofseveral freshly cut subsamples.
Fungaltotalgenome
3 Birds Feather DNA extraction of thearchaeological feather followedthe same protocols as the freshsamples. The protocols followsstrict contamination-control forthe analysis of ancient remains.Two and five feather barbs wereremoved from the feather shaft.Barbs were first rinsed in 3%sodium hypochlorite for 30seconds to remove possiblesurface contamination, then
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page 72 / 107
4 Cow Milkandcurd
Total DNA was isolated from milksamples by a phenol/chloroformmethod, followed by ethanolprecipitation according toSambrook et al.(1989)Cheesesamples were cut into smallpieces, and total DNA
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Totalgenome -fragmentedgenome
page 73 / 107
from cheese matrix was obtainedby performingphenol/chlorophorm as describedabove.
DNA from reference strain ofbacteria were prepared from pureculture according to theguanidium extraction proceduredescribed by Pitcher et al. (1989).
Totalgenome,includemicrobialgenom
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page 74 / 107
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5
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page 76 / 107
Clams
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Muscletissue
page 77 / 107
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Individuals with opened andclosed shells were preservedfromeach beach in 80% ethanol.Muscle tissue was extracted fromthe middle and apex region (c.1mm2) of the foot and cleanedwith ethanol (75%) to removesand, detritusor external organicmatter.DNA extraction wasperformed with the QiagenDNAMini kit, with slighltymodification to increase theconcentration of DNA, i.e. weused 150 µl of elution buffer.
page 78 / 107
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Fragmentof themitochondrial COIgene
totalgenome
page 79 / 107
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6
page 80 / 107
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page 81 / 107
Seacucumber
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Oraltentacle
page 82 / 107
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Upon collection, specimens werecleaned in seawater, immediatelyfrozen on dry ice (-70°C).Typically 200 mg of oral tentaclewas ground in liquid N2 in thepresence of 600 µl of proteinasesolution (50 mM Tris–HCl, pH 7.5,50 mM EDTA, pH 8.0, 0.4% SDS,0.5 mg/ml Proteinase K). Theground samples wereimmediately placed at 65°C forminimum of 2 h. The digestedsamples were repeteadlyextracted withphenol:chloroform:isoamylalcohol, 25:24:1. The resultantaqueous phase was adjusted to0.5 M NaCl and to 1%cetyltrimethylammonium bromide(CTAB) and incubated for a further20 min at 65°C. After two finalphenol/chloroform extractions,total DNA was precipitated byadding an equal volume ofisopropanol followed bysedimentation at 13,000 rpm for20 min. The DNA pellet was rinsedwith 500 µl of 70% ethanol,air-dried, and resuspended in 200µl of TE buffer (10 mM Tris–HCl,pH 7.5, 1 mM EDTA).
page 83 / 107
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16S
mitochondrialribosomalDNAfragments
totalgenome
page 84 / 107
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page 85 / 107
7
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Snake
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0C.Sloughed skins, mostly collectedat the entrance of identified adderdomains, were preserved dry atroom temperature for up to twoyears. An additional fresh sloughwas obtained from an adderobserved in the process ofecdysis. Snake faeces (associatedwith sloughs) collected from arange of UK sites were eitherimmediately frozen at -20oC orpreserved in 95% ethanol. A 10 µlblood sample, obtained by caudalextraction from an adder andstored in 90 µl of Seutin's buffer(Seutin et al.,1991) at roomtemperature, was collected as apositive PCR control.Sloughedskin required a rehydration stepto remove impurities prior to DNAextraction. A fragment (1-2 cm2)of slough was rinsed in 1 ml ofddwater at 55 oC in a rockingincubator for 4-6 h, and repeatedfor a further 8-12 h. DNAextraction was performed onthese rehydrated samples, eggyolks (approximately 0.2 cm3),NIS muscle (1 cm3) and blood (5µl in Seutin'sbuffer) usingQiagenDNeasy tissue extractionkit. Faecal material was extractedusing QIAamp@ DNA stool minikit.
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TM (YasuiKikai,Osaka, Japan). The nailpowder was lysed in a urea-lysissolution (2 M urea; 0.5 %SDS; 10mM Tris-HCl, pH 7.5; 0.1 M EDTA)containing 1 mg/ml proteinase Kand 40 mM DDT at 55 °Covernight. Nail DNA was extractedwith phenol/chloroform, andprecipitated with ethanol andsodium acetate. Precipitated nailDNA was dissolved again inextraction buffer (0.5 % SDS; 10mM Tris-HCl, pH 7.5; 0.1 M EDTA)containing 1 mg/ml proteinaseK, and incubated at 55 °Covernight. DNA was purifiedagainas above, and was suspended in30 µl of 1x TE buffer.
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