Datp → HCID 86612373, compound

Temperature

65 °C

A second treatment of the DNA with the Klenow fragment of E. coli DNA polymerase I was conducted as described above except that 0.05 mM deoxycytidine triphosphate was substituted for the 0.05 mM deoxyadenosine triphosphate of the previous reaction. This produced a fragment with the sequence ##STR19## near the beginning of the rennin gene. After phenol extraction, ether extraction, and ethanol precipitation, the DNA was redissolved in water and treated with S1 nuclease (Boehringer/Mannheim, 100 units) for 30 minutes at room temperature i a 50 μl reaction containing Buffer S. This enzyme removed the 5' single-stranded DNA from the fragment leaving the sequence ##STR20## Thus, the beginning of the rennin sequence is at the 5' end of the DNA fragment. A synthetic oligonucleotide containing a ClaI restriction site and ending with the nucleotides ATG (i.e. CATCGATG, Collaborative Research, Inc., 5 μg) was kinased with Y32 -P-ATP using T4 polynucleotide kinase ((P-L Biochemicals, 3 units) for 30 minutes at 37° C. in a 25 μl reaction containing Buffer Y. This kinased linker (about 200 pmoles was ligated to the treated DNA fragment (about 5 pmoles) by incubation with T4DNA Ligase (N.E. Biolabs, 900 units) at 15° C. overnight in Buffer L. The reaction was terminated by heating at 65° C. for 5 minutes. Four microliters of 10x ClaI buffer (1x=10 mM Tris·HCl pH 8, 10 mM MgCl2), and 10 μl of restriction endonuclease ClaI (Boehringer/Mannheim, 27 units) were added. The resulting mixture wax incubated at 37° C. for one hour. Four microliters were removed for analysis on a polyacrylamide gel, followed by the addition of 1 μl of 10x ClaI buffer, 10 μl of ClaI enzyme, and 3 μl water. This mixture was incubated for an additional hour. The treated DNA containing the desired rennin sequences was purified by separation in a 2% agarose gel containing 40 mM Tris·acetate buffer pH 8.3. The DNA was visualized by long wave ultraviolet irradiation and removed from the gel by the freeze-thaw method described above. This fragment was then ready for insertion into the appropriate vector.

1. customThis produced a fragment with the sequence ##STR19## near the beginning of the rennin gene
2. extractionAfter phenol extraction, ether extraction, and ethanol precipitation
3. dissolutionthe DNA was redissolved in water
4. additiontreated with S1 nuclease (Boehringer/Mannheim, 100 units) for 30 minutes at room temperature i a 50 μl reaction
5. additioncontaining Buffer S
6. customThis enzyme removed the 5' single-stranded DNA from the fragment
7. customleaving the sequence ##STR20## Thus, the beginning of the rennin sequence
8. customfor 30 minutes
9. customat 37° C.
10. customin a 25 μl reaction
11. additioncontaining Buffer Y
12. customThe reaction was terminated
13. additionFour microliters of 10x ClaI buffer (1x=10 mM Tris·HCl pH 8, 10 mM MgCl2), and 10 μl of restriction endonuclease ClaI (Boehringer/Mannheim, 27 units) were added
14. customFour microliters were removed for analysis on a polyacrylamide gel
15. additionfollowed by the addition of 1 μl of 10x ClaI buffer
16. waitThis mixture was incubated for an additional hour
17. customThe treated DNA containing the desired rennin sequences was purified by separation in a 2% agarose gel
18. additioncontaining 40 mM Tris·acetate buffer pH 8.3
19. customThe DNA was visualized by long wave ultraviolet irradiation
20. customremoved from the gel by the freeze-thaw method

ORD record

ord-ebada0ee8ba04e08915e658042b4c3db

Source dataset

uspto-grants-1996_03

DOI

10.6084/m9.figshare.5104873.v1

Patent number

US05496711