| |
1. Digest the plasmid with either Bal 31 or ExoIII and S1 Nuclease to create a set of deletion mutations in the region of interest.
2. Stop the digestion reaction by either heating to 60°C or adding EDTA to a final concentration of 50 mM.
3. Precipitate the DNA by adding 2.5 volumes of 100% Ethanol.
4. Ligate the linker to the DNA and digest the construct with a Restriction Enzyme (see Hint #1).
5. Run an Agarose Gel to isolate the fragments (see protocol for Agarose Gel Electrophoresis of DNA).
6. Ligate the fragments to the vector fragment creating an intact plasmid.
7. Transform E. coli with the plasmid.
8. Pick transformed colonies and isolate the DNA plasmids using a miniprep (see protocols for Plasmid Minipreps).
9. Digest the plasmid DNA with a Restriction Enzyme to create a terminus at a deletion endpoint.
10. Determine the Restriction Enzyme fragment sizes by nondenaturing polyacrylamide gel electrophoresis (see protocol for Nondenaturing PAGE; see Hint #2).
11. Sequence the collection of fragments which span the region of interest.
12. Design complimentary oligonucleotides between the linker and a nearby restriction site.
13. Prepare the fragments for ligation to the oligonucleotides. Fragment number 1 should span the linker site and promoter fragment. Fragment number 2 should span the promoter region and a restriction site near the scanning region. Isolate the fragments.
14. Prepare the Oligonucleotides at 75 to125 μg/ml in TE/NaCl Buffer.
15. Combine equal amounts of each Oligonucleotide and heat the mixture to 65°C for 10 min.
16. Slowly cool the mixture to room temperature and determine the final DNA concentration.
17. Check to make sure that the Oligonucleotides have hybridized.
18. Combine equal amounts of fragment number 1 and fragment number 2. Add about 50-fold excess of the hybridized Oligonucleotides relative to the plasmid fragment. Attempt to get a final DNA concentration of about 40 μg/ml.
19. Ligate the DNA using T4 Ligase at 15°C for 2 hr.
20. Using TE buffer, dilute the sample to about 1 μg/ml.
21. Heat the diluted sample to 65°C for 10 min, then slowly cool to it room temperature.
22. Transform E. coli using 20 to 25 μl of the ligation reaction and plate out the bacteria.
23. Pick transformed colonies and isolate the plasmid DNA using a miniprep.
24. Check the fragments by restriction digest and DNA sequencing.
|
| 0.5 mg/ml Bovine Serum Albumin |
 |
| T4 DNA Ligase Buffer (10X) |
| 50 mM DTT
0.5M Tris, pH 7.5
50 mM MgCl2
|
|
| TE/NaCl Buffer |
| 10 mM Tris
1 mM EDTA
pH 8.0
150 mM NaCl
|
|
| 0.5 M EDTA |
| pH 8.0
|
|
|
| |
DNA Ligase, T4
Restriction Enzyme
S1 Nuclease
Tris
ExoIII
Bal 31
Oligonucleotide
Ethanol
EDTA
Magnesium Chloride
DTT
Sodium Chloride
Bovine Serum Albumin (BSA)
|
| |
1. Construct the DNA such that the fragment has a linker at the deleted end and the vector at the other end.
2. You will need fragment sizes between 175 bp and 300 bp to accurately determining the location of each mutation.
|
