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| Large genes can be cloned by identifying overlapping cosmid clones. This protocol describes digestion of the cosmid insert to remove all but the ends of the insert. The cosmid is then recircularized and the new plasmid is used as a probe to identify other cosmid clones containing adjacent sequence. In this way, one can "walk" down the chromosome. |
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1. Digest an aliquot of miniprep DNA of a positive cosmid clone to completion by incubating 0.5 to 1.0 μg cosmid DNA with an appropriate restriction enzyme that cleaves several times within the insert but not within the vector sequence itself (or see Hint #1) in a 50 μl reaction volume (see Hint #2).
2. Run half of the reaction on an Agarose gel to make sure that the digestion is complete (see Protocol on Agarose Gel Electrophoresis of DNA).
3. Heat inactivate the enzyme if possible by heating the reaction to 65°C for 20 min and go to Step #7. If the enzyme is heat stable, bring the reaction volume to 50 μl using ddH2O, add 0.5 μl of Diethylpyrocarbonate Solution.
4. Incubate at 37°C for 20 min.
5. Add 2.5 μl of 1 M Tris Buffer to inactivate the Diethylpyrocarbonate.
6. Incubate at 65°C for approximately 10 min (see Hint #3).
7. Bring the reaction volume (or collected fraction from spin column) to 100 μl with Ligase Buffer and 100 Units of T4 DNA Ligase so that the final concentration of Ligase Buffer is 1X.
8. Incubate at 16°C overnight to recircularize the digested cosmid vector.
9. Transform competent bacteria cells with the ligation mix (see Protocol on Transformation of E. coli).
10. The resulting endclones can be hybridized to cosmids from the previous step in the chromosome walk to identify overlapping clones that contain adjacent sequence (see Protocol on Screening of Cosmid Libraries and Isolation of Positive Colonies). Endclones can also be used for in situ hydridization to polytene chromosomes to get an accurate picture of the progress of the walk.
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| Ligase Buffer (10X) |
| 10 mM ATP
0.5 M Tris-Cl, pH 7.6
100 mM MgCl2
250 μg/ml BSA, Fraction V (Sigma)
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| 1 M Tris Buffer |
| 1 M Tris-Cl, pH 8.0
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| Diethylpyrocarbonate Solution |
| Prepare in 100% Ethanol
10% (v/v) Diethylpyrocarbonate (CAUTION! see Hint #4)
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Tris
Ethanol
ATP
Restriction Enzymes
DNA Ligase, T4 Bacteriophage
Magnesium Chloride
Diethyl Pyrocarbonate (DEPC)
Bovine Serum Albumin (BSA), Fraction V
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1. Many cosmid libraries are constructed using vectors where end fragments from either end of the cosmid can be easily subcloned. For example, the cosPneo vector has a polylinker containing a BamHI site flanked on one side by an EcoRI site and on the other side an XbaI site. If the cosmid library in this example is made by inserting genomic DNA into the BamHI site, then one end of the cosmid can be isolated by an intramolecular ligation of a complete XbaI digest and the other cosmid end isolated by an intramolecular ligation of a complete EcoRI digest.
2. Isolate cosmid end fragments as rapidly as possible when chromosome walking.
3. Optional. To ensure high cloning efficiency, the digest can be run over a Sephadex G50 (P10) spin column.
4. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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