Primer Extension

1. To0.5pmoleprimer(0.5µl10pmoles/µl)add1µl10xT4polynucleotidekinasebufferfromRochekit.
2. Add2.5µl³²P-γ-labelleddATP(5µlifusing³²P-γ-labelleddATPpastitsfirsthalflife).
3. AddDEPCtreatedwatertomakeupto9µl(4.5µlifusing10pmoles/µlprimer).
4. Add1µlT4polynucleotidekinasefromsamekit.
5. Incubatefor30minutesat37°C.
6. RemovetopandbottomcapfromNAP-5column(Amersham-PharmaciaBiotech)todrainofstorageliquid.
7. FillupwithDEPCtreatedwater3timesandallowtodrain.
8. Add500µlDEPCtreatedwatertoprimerlabelingreaction.
9. AddprimerlabelingreactiontoNAP-5columnandallowcoldflowthroughtodrainoff.
10. Add1mlofDEPCtreatedwaterandcollecteluatewithpurifiedhotprimer.

    ReversetranscriptionwithSuperscriptIIfromradioactiveprimer

11. Take30µgofRNAforhighlyexpressedgenesor50µgforpoorlyexpressedgenesandadjustvolumeto40µl.
12. Add60µloftheeluatefromthepurificationoflabeledprimer.
13. Add10µl(1/10volume)of3Msodiumacetate.
14. Add250µlof95%ethanol.
15. Incubateovernightat-70°C.
16. Spinfor30minutesatfullspeed.
17. Removeanddiscardsupernatantcarefully.
18. Add500µl75%ethanol.
19. Spin10minutesatfullspeed.
20. Removeanddiscardsupernatantcarefully.
21. Resuspendin30µlofDEPCtreatedwater.
22. Incubateat90�Cfor3minutes.
23. Coolslowlytounder30°C.
24. Add18.5µlofDEPCtreatedwater.
25. Add20µlof5*SuperscriptIIbuffer.
26. Add10µl0.1MDTT.
27. Add20µldNTP-mixforprimerextension.
28. Add1µlofRNasin(RNase-inhibitor,Promega).
29. Add0.5µlSuperscriptIIreversetranscriptase.
30. Incubateat42�Cfor90minutes.
31. Add1µl0.5MEDTAandmix.
32. Add0.5µlRNaseA(Promega)andmix.
33. Incubateat42°Cfor10minutes.

    PurificationbyPhenol/Chloroformextraction

34. Add100µlPhenoltothereaction,mixbyvigerousshaking.
35. Spin10minutesatfullspeedtoseparatephases.
36. Transfertheaquaous(top)phasetoanewtubewith125µl50:50Phenol:Choloroform.
37. Add25µldH₂OtothetubecontainingthePhenolphase.
38. TransfertheaquaousphasetothetubewithPhenol:Chloroform,mixbyvigerousshaking.
39. Spin10minutesatfullspeedtoseparatephases.
40. Transfertheaquaous(top)phasetoanewtubewith150µlCholoroform.
41. Add25µldH₂OtothetubecontainingthePhenol:Cholorofomphase.
42. TransfertheaquaousphasetothetubewithChloroform,mixbyvigerousshaking.
43. Spin10minutesatfullspeedtoseparatephases.
44. Transfertheaquaous(top)phasetoanewtube.
45. Add25µldH₂OtothetubecontainingtheChloroformphase.
46. Transfertheaquaousphasetothetubewiththeaquaousphasefrompreviously.
47. Add17.5µl(1/10thvolume)3MSodium-Acetateandmix.
48. Add550µl(3volumes)95%ethanol.
49. Leaveat-70°Cfor15minutesorovernight.
50. Spin30minutesatfullspeed.
51. Removesupernatant,andadd500µl75%ethanol.
52. Spinfor10minutes.
53. Removesupernatantandairdryuntilnoethanolisleftintube.
54. Dissolvein6µldH₂O.
55. Add4µlstopsolutionfromAmershammanualsequncingkit.

    Loadandrunonamanualsequencinggelalongwithamanualsequencingreactionpreferablydoneontherelevantgenewiththesameprimeraccordingtomanufactureresdirections.DrythegelandexposeX-rayfilmtoitfor1to2days.

ProductionandpurificationoflabeledcDNA

dNTP-mixforprimerextension

10µl100mMdATP,10µl100mMdCTP,10µl100mMdGTP,10µl100mMdTTP,10µlDEPC-treatedwater.