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DMS Chemical Footprinting of RNA188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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DMS Chemical Footprinting of RNA

Notes:

  1. RNAshouldbeat1uMconcentration(1pmole/uL)
  2. Thereare3conditions(useablank/backgroundreactionforeachcondition).

Solutions

CEBuffer(10X)

  • 1MNaCacodylate(4.28ginto20mLH2O)pH7.4
  • 5mLof1MNaCacodylate
  • 100uLof0.5MEDTA
  • 44.9mLH2OSterilefilterandallowvacuumtocontinue5mins.toallowfordegassing.

1XCEBuffer

  • 1mL10XCE
  • 9mLH2O

DMSSolution:

  • 15uL100%ETOH
  • 3uLDMS

QuenchSolution:

  • 1.4mL2-beta-mercaptoethanol
  • 0.5mL3MSodiumAcetate(AmbionBufferkit)
  • 3.1mLH2O

*DiluteRNAto1uMin1xCEbuffer

Conditions

Condition1:

10uLRNA(1uMconcentration)2.5uL10xCEbuffer12.5uLH2O

Condition2:

10uLRNA(1uMconcentration)2.5uL10xCEbuffer1.25uL2MKCl(AmbionBufferkit)11.25uLH2O

Condition3:

10uLRNA(1uMconcentration)2.5uL10xCEbuffer1.25uL2MKCl(AmbionBufferkit)*1.25uL100mMMgCl2(dilutedfrom1MMgCl2AmbionBufferkit)10uLH2O*DONOTADDMgCl2UNTILREADYTOINITIATEFOLDING.

Procedures

FoldingSteps

  1. Heatthe25ul(Mgfree)reactionmixat90Cfor3mins.
  2. Takethesampleoutandletitcooltoroomtemperatureforabout10-15mins-,afterwhichgiveaquickspinonatabletopcentrifuge.
  3. Putthesampleina50Cheatingblockandletitequilibriateat50Cfor10mins.
  4. AddMgCl2(finalMgconcentration10mM)tosamples.
  5. Placesampleina50Cheatingblockfor15-30mins.
  6. Takethesampleoutandputitinaheatingblocksetatthefoldingtemperature(25Cor37C).Incubateatthefoldingtemperaturefor1hour.
  7. Initiatemodificationreaction(OHfootprinting/DMS....etc.)atthesametemperatureusedforfolding(25Cor37C).
  8. Similarly,wepreparetheunfoldedsampleinanidenticalwaybutskiptheadditionofMgCl2atstep3.

DMSmodification

  1. Tothe12.5uLoffoldedRNA,add0.5uLofDMSsolution
  2. Incubatefor2minutesat25Cor37C.
  3. Add475uLofquenchsolution(eventosampleswithoutDMSmod)
  4. Add1mLof100%ETOH
  5. FreezeO/Nat-80C
  6. PrepareRNAforRT

ReverseTranscription

  1. Spindownsamplesat14,000RPMat4Cfor1hour
  2. Removesupernatant
  3. Rinsewith100uLof70%ETOH
  4. Spinat14,000RPMat4Cfor0.5hours
  5. Removesupernatantanddrypelletinspeed-vac,mediumheatfor3mins.

RTBuffers

AnnealingBuffer(50mMTris-CL,pH8.3,60mMNaCl,and10mMDTT)

Tris-Cl5uL
NaCl1.1uL
DTT10uL
H2O83.9uL
Totalvolume100uL

ReverseTranscriptionMix

5xFSbuffer4uLperrx
0.1mMDTT1uLperrx
RNaseOut2uLperrx
10mMdNTPmix2uLperrx
*ddNTP5mM5uLperrx
SuperscriptIIIenzyme1uLperrx

*Onlyuseifdoingadideoxysequencingreaction.

RTReaction

  1. ResUSPendRNApelletin9uLofannealingbuffer
  2. Add1uLofcy5’labeledprimerat10uM
  3. Heatto90Cfor2min.andslowlycoolto25Ctoallowprimertoanneal.(1-1.5hours)Note:Keeptubesinheatblockandpulloutwholeblockfromheat.UseaThermometertomonitorthetemp.
  4. Add9uLofreversetranscriptasemix
  5. Heatsolutionat55Cfor5mins
  6. THENadd1uLofsuperscriptIIIenzymetoeachsample.
  7. Mixgently,brieflycentrifuge,incubateat55Cfor10-15mins.

CleanUpSample

  1. Add2uLof2NNaOH
  2. Incubateat95Cfor3mins.
  3. Add2uLof2NHCltoneutralize
  4. Add3uLof3MNa-acetate
  5. Add1uLof100mMMgCl2
  6. Add90uLof100%ETOH
  7. Centrifugeat14,000RPMfor30minsat4C
  8. Removesupernatant
  9. Add50uL70%ETOH
  10. Centrifugeat14,000RPMfor30minsat4C
  11. Drypelletandre-suspendin60uLSampleLoADIngSolution(Beckman)
  12. Load60uLofsamplein96wellplate
  13. Add1uLofLaddertoeachwell
  14. PlacedropofmineraloilontopandfollowCEQprotocol


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