First strand of cDNA synthsis

1.Add 2 ul of Not I primer-adapter to a sterile 1.5 ml RNAase free tube, add 4.5 ul mRNA of molt4 cells (~5.2 ug), add 0.5 ul DEPC-H2O

2. Heat the mixture at 70oC for 10 min, and quick chill on ice, collect the contents of the tube by brief centrifugation.

3. Add the following component : 4 ul 5X first strand buffer
2 ul 0.1 M DTT
1 ul 10 mMdNTP
1ul {a-32P}dCTP(1uCi/ul, 800 uc /mmol)
mix by tapping the tube with a finger, and briefly spin it to collect the reaction

4. Add 5ul SuperScript II RT, mix again and spin the tube

5. Incubate it at 37oC for 1 hour.

6. Place the tube on ice to terminate the reaction

7. Remove 2 ul from the reaction , and add it to a microcentrifuge tube containing 43 ul of 20 mM EDTA(pH8.0) and 5ul of yeast tRNA, use it to calculate the effiency of the first strand synthesis.

8. On ice, add the following components:
DEPC-treated water 93ul
5xsecond strand buffer 30
10 mM dNTP mix: 3 ul
E.coli DNA ligase(10U/ul) 1
E.COli DNA polymerase I(10U/ul) 4
E.coli Rnase H(2U/ul) 1
Final volume 150ul
Mix them well by tapping, incubate it at 16oC for 2 hrs, do not let the temperature go beyond 16 oC.

9. Add 2 ul (10 units ) of T4 DNA polymerase to the reaction, and continue incubatinf at 16 oC for additional 5 min

10. Place the reaction on ice, and add 10 ul of 0.5M EDTA, remove 10 ul from the reaction, and add it to a 1.5 ml tube containing 35ul of 20 mM EDTA(pH7.5) and 5 ul of yeast tRNA. This mixture will be used in ca;culating second strand yield.

11. Add 0.5 ml PN buffer from Qiegen Nucleotide Removal Kit to the remaining reaction, and mix it ,load it into the spin column, and follow the procedures comimg with the kit to purify the double strand DNA.

12. Elute the cDNA from the column with 25 ul of sterilized water, and add 5xT4 DNA ligase buffer 10 ul, EcoRI-SmaI adapter(1ug/ul, Stratagen) 10 ul , T4 DNA ligase , mix gently, and incubate t he reaction at 16oC for 18 hours.

13. Add 0.5 ml of PN buffer to purify the DNA

14. Elute DNA from the column with 4 ul of water. add reaction 3 buffer 5ul , Not I 4ul, mix and incubate it for 2 hours at 37 oC.

15. Inactivated the enzyme by heating at 65oC for 15 min .

16. Size select the cDNA by column chromatography:

1). Add 40 ul TEN buffer to the above heated reaction, place it on ice.

2). Drain a column, pipette 0.8 ml of TEN buffer onto the upper frit, and let it drain completely. Repeat this step three more times to remove the ethanol in the column completely.

3).Add the entire cDNA sample to the center øf the top frit and let it drain into the bed. Collect the effluent into tube 1.

4) Add 100 ul of Ten buffer to the column, and collect the effluent intotube 2.(Note: Let the column drain completely before the addition of new 100ul aliquøt.

5). Beggining with the next 100-aliquot of TEN buffer, collect single drop fractions into individual tubes. Continue adding 100 ul aliquots of TEN buffer until you collect a total of 18 drops into tubes 3 through 20, one drop per tube.

6) Using an automatic pipette, measure the volume in each tube; use a fresh tip for each fraction to avoid cross contamination.

7) Place the remaining tubes in a scintillation counter , and obtain Cerenkov counts for each fraction . Count the entire sample in the tritium channel without scintillation liquid.(!).

8) Measure the DNA concentration of each fraction in which the Cerenkov counts exceed background by Dip DNA kit from Invitrogen:

9).Collect cDNA from tubes 8, 9, 10, 11, and keep them individually at -70 oC to be checked for the size and cloned into a suitable vector.