Nested Deletions Using Exonuclease-III and Mung Bean Nuclease
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A nested series of deletions can be produced by: cutting plasmid DNA (2.5ug per time point, see below) with an enzyme leaving a blunt (eg EcoRV) or 5'' overhand (eg EcoRI) on the side that the deletions are to proceed. To prevent deletions in the other direction cut with an enzyme leaving a 3'' overhang (eg. Kpn. NOTE: that SacII will not work for this purpose) or cutting with a 5'' overhang creating enzyme and filling with thionucleiotides as follows. This is more reliable than using a 3'' overhang:
- Cut with the enzyme to be filled in
- Heat 70° C for 15 min. Quench on ice.
- Add thio-d NTP to 40uM and 5U klenow
- 20 min at RT. Heat to 65°C for 10min.
- Phenol/CHCL3 extract and ethanol ppt.
- Cut with the second enzyme on the side that deletions are to proceed in around 100ul. Heat inactivate the enzyme when complete. A linearized clone and linearized vector (1ug ea.) provide useful markers for the start and end of the deletion series, respectively, when the deletions are run on a gel.
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Resuspend the DNA in
- 1X exo buffer (see end) - 10mM 2-mercaptomethanol (prepared fresh from 14M stock) - Xul DDW - The volume should equal 25ul per time point. Heat to 30° for 5 min to equilibrate at incubation temp. - Dispense 20ul 10X mung bean buffer, 155ul DDW into eppendorf tubes for each time point. - Have on hand crushed dry ice. - Add 20U of ExoIII per ug of DNAand vortex.
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Remove 25ul aliquot’s into the diluted mung bean buffer at Xsec intervals according to the following table and place on dry ice.
- 37° = 400bp/min - 34° = 375bp/min - 30° = 230bp/min - 23° = 125bp0/min -
I generally do my incubations at 30°C and take my first aliquot at 20 sec and thereafter at 40-80 sec intervals (a test for the best timepoints may be useful). - When finished remove tubes from dry ice and heat to 68°C for 15 min then place on ice.
- To make the DNA blunt ended, add 7.5U mung bean nuclease (diluted in mung bean dilution buffer, see end) per 2.5ug time point and incubate at 30° for 30 min.
- Ethanol ppte, resuspend and load onto a 0.8% TAE gel. Run and excise bands free from undigested DNA. Isolate DNA and ligate overnight. Transform and pick 2-4 colonies per time point for mini gel analysis and subsequent sequencing.
Buffers
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2X exo buffer
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10X mung bean buffer
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1X mung bean dilution buffer
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Comments :
- The DNA must be >85% supercoiled. ExoIII will digest from nicks. Use CsCl purified DNA and avoid using the UV lamp to isolate plasmids from CsCl gradients. Check the DNA on a gel vs. cut plasmid.
- The DNA must be fully restricted with the 5'' overhang (otherwise background of undigested DNA) and with the 3'' overhang (otherwise deletions proceeds in both directions).
- Use a gel to isolate the deletion fragments rather than simply extracting the DNA (Extraction of the deletions after the mung bean step requires the addition of 4ul 20%SDS, 10ul 1M Tris pH 9.5, 20ul 8M LiCl and 250ul phenol/CHC13 , followed by re-extraction and precipitation) after the mung bean step. Gel purification is preferable as it removes the background and allows you to monitor how well the reaction has proceeded.