cDNA synthesis using superscript II

ATTENTION:Thisisexpensive.Onereactionasdescribedhereisca.6Euro!CheckthequalityofyourRNAbygeltobesureitisnotdegraded.

1. Preparetheannealingmix:

   RNA	50ng/µl	1µg
   randomhexamers(100ng/µl)	25ng/µl	5µl
   water		addto20µl

2. Annealtheprimersinathermocycler:

   1.	70°C	10min
   2.	25°C	10min
   3.	4°C	store

3. Preparetheenzymemix:

   5xFirstStrandBuffer	8µl
   DDT	4µl
   dNTP(10mMeach)	2µl
   SUPERaseinhibitor	1µl
   SuperScriptII	1µl
   water	4µl

   Preparethismixasamastermixandaddthen20µltoeachreaction.
4. SynthesizethecDNAinathermocycler:

   1.	25°C	10min
   2.	37°C	45min
   3.	42°C	45min
   4.	70°C	15min
   5.	4°C	store

5. DilutethereactionmixforuseinqPCRAdd160µlwatertothe40µlreactionmix.Use2µlperqPCRreaction(thatsequivalentto10ngRNA).

RandomPrimerHexamers:

Dilute33.3µlRandomPrimerHexamers(3µg/µl)with966.7µlwatertoobtaina100ng/µlsolution.

SuperScriptII(#18064-014)byInvitrogenRandomPrimerHexamers(3µg/µl(#48190-011)byInvitrogenSUPERaseInhibitor(#2694)byAmbion

1.Preparetheannealingmix:

RNA	50ng/µl	1µg
randomhexamers(100ng/µl)	25ng/µl	5µl
water		addto20µl

Insteadoftherandomhexamersyoucanuse4pmolegene-specificprimersor1µgOligo(dT)_12-18(eachgivenastotalamountforthisreactionmix).Iprefertherandomhexamers,becauseonesynthesiscanbeusedforwhateveryouwanttodetectandisnotenrichingthe3endsoftheRNA.Furthermore,itjustworkswell.

2.Annealtheprimersinathermocycler:

1.	70°C	10min
2.	25°C	10min
3.	4°C	store

3.Preparetheenzymemix:

5xFirstStrandBuffer	8µl
DDT	4µl
dNTP(10mMeach)	2µl
SUPERaseinhibitor	1µl
SuperScriptII	1µl
water	4µl

Preparethismixasamastermixandaddthen20µltoeachreaction.Thisworksreallywell.ImakeabigmixandaddittoeachtubeassoonasthePCRisfinished.Ijustdothisatroomtemperaturetoavoidthetroublewithice.

4.SynthesizethecDNAinathermocycler:

1.	25°C	10min
2.	37°C	45min
3.	42°C	45min
4.	70°C	15min
5.	4°C	store

5.DilutethereactionmixforuseinqPCR

Add160µlwatertothe40µlreactionmix.Use2µlperqPCRreaction(thatsequivalentto10ngRNA).ForsomeapplicationsitisnecessarytogetridoftheRNA.UseaRNaseincubationfollowedbyacleanupusingspincolumns.

• TheexpensivestuffistheSuperScriptenzyme.Therestissuppliedinsufficientamounts.Ifyouhavelessthan1µgRNA,youcanconsidertoreducethevolumetohalfandtheenzymetomatchtheamountofRNA.Beforeyouprocesshundredsofsamplestryonesamplewithyouroptimizedprotocolversusthisstandardversion.