Nested Deletions using exonuclease-III and mung bean nucleas

Friday, November 21, 2003

Description
Nested Deletions using exonuclease-III and mung bean nuclease

Procedure
ExoIII has a 3' to 5' exonuclease activity which must initiate on blunt ended DNA or DNA with a 5' overhang.

1. A nested series of deletions can be produced by: cuting 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:

a) Cut with the enzyme to be filled in

b) Heat 70 C for 15 min. Quench on ice.

c) Add thio-d NTP to 40uM and 5U klenow

d) 20 min at RT. Heat to 65C for 10min.

e) Phenol/CHCL3 extract and ethanol ppt.

f) 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.

2. 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 equibrate at incubation temp.

Dispense 20ul 10X mung bean buffer, 155ul DDW into eppendeorf tubes for each time point.

Have on hand crushed dry ice.

3. Add 20U of ExoIII per ug of DNAand vortex.

4. Remove 25ul aliquots 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 30C and take my first aliquot at 20 sec and thereafter at 40-80 sec intervals (a test for the best timepoints may be useful).

5. When finished remove tubes from dry ice and heat to 68C for 15 min then place on ice.

6. 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.

7. 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 (over)

2X exo buffer

100mM Tris pH8

10mM MgC12

20ug/ml tRNA

10X mung bean buffer

300mM NaAC pH 5

500mM NaCl

10mM ZnC12

50% glycerol

1X mung bean dilution buffer

10mM NaAC pH 5

0.1 mM ZnC12

1mM cysteine

0.1% triton X-100

50% glycerol

Comments:

1. 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.

2. 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).

3. 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.

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Nested Deletions using exonuclease-III and mung bean nucleas