Wiese Lab:Plasmid DNA Miniprep

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(New page: Back to Protocols ==Plasmid DNA Miniprep== This procedure is used when many transformants need to be screened. The reagents are much less expensive than the mini...)
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#Transfer a single bacterial colony into 2 mL of LB containing the appropriate antibiotic in a loosely capped 15-mL tube.  Incubate the culture overnight at 37C with vigorous shaking.
#Transfer a single bacterial colony into 2 mL of LB containing the appropriate antibiotic in a loosely capped 15-mL tube.  Incubate the culture overnight at 37C with vigorous shaking.
#Pour 1.5 mL of the culture into a microfuge tube.  Centrifuge at (12,000g for 30 sec, or 5,000g for 5 min) at (4C or RT) to pellet the bacteria. <i>(pellets can be frozen here)</i>
#Pour 1.5 mL of the culture into a microfuge tube.  Centrifuge at (12,000g for 30 sec, or 5,000g for 5 min) at (4C or RT) to pellet the bacteria. <i>(pellets can be frozen here)</i>
-
#Remove the medium by decanting and inverting tube on paper towel, or by aspiration.  
+
#Remove the medium by decanting and inverting tube on paper towel, or by aspiration.
 +
 
#Resuspend the pellet in 200 μl of ice-cold Solution I. <i>(tubes can be stored cold for a few hours here)</i>
#Resuspend the pellet in 200 μl of ice-cold Solution I. <i>(tubes can be stored cold for a few hours here)</i>
#Add 400 μl of freshly prepared Solution II.  Mix contents of the tube by inverting the tube rapidly five times.  Store the tube on ice for 5 min.
#Add 400 μl of freshly prepared Solution II.  Mix contents of the tube by inverting the tube rapidly five times.  Store the tube on ice for 5 min.
Line 17: Line 18:
#Precipitate the DNA (add 500 ul isopropanol <i>(tubes can be stored cold ON here)</i>; leave at RT 15 min; spin 5 min at 12K; drain; wash by centrifuging (12K for 5 min) with 500 ul 70% EtOH; drain; dry) and resuspend in 30 μl of TE (pH 8) <i>(can be resuspended ON)</i>.  TE may be prepared to contain RNase A at 20 ug/ml by adding 1 ul RNase stock (10mg/ml) to 500 ul TE. TE is 10 mM Tris pH 8.0 + 1 mM EDTA pH 8.0.
#Precipitate the DNA (add 500 ul isopropanol <i>(tubes can be stored cold ON here)</i>; leave at RT 15 min; spin 5 min at 12K; drain; wash by centrifuging (12K for 5 min) with 500 ul 70% EtOH; drain; dry) and resuspend in 30 μl of TE (pH 8) <i>(can be resuspended ON)</i>.  TE may be prepared to contain RNase A at 20 ug/ml by adding 1 ul RNase stock (10mg/ml) to 500 ul TE. TE is 10 mM Tris pH 8.0 + 1 mM EDTA pH 8.0.
-
=====Solution I=====
+
=====Solution I=====
-
50 mM glucose <br>
+
50 mM glucose <br>
-
25 mM Tris*Cl (pH 8.0) <br>
+
25 mM Tris*Cl (pH 8.0) <br>
-
10 mM EDTA (pH 8.0) <br>
+
10 mM EDTA (pH 8.0) <br>
=====Solution II=====
=====Solution II=====
-
0.2 N NaOH <br>  
+
0.2 N NaOH <br>
 +
 
1% SDS <br>
1% SDS <br>
 +
{| cellspacing="0" cellpadding="0" border="1"
 +
| Component
 +
| < 5 samples
 +
| < 10 samples
 +
| < 15 samples
 +
| < 20 samples
 +
|-
 +
| 10% SDS
 +
| 0.2 ml
 +
| 0.4 ml
 +
| 0.6 ml
 +
| 0.8 ml
 +
|-
 +
| 1N NaOH
 +
| 0.4 ml
 +
| 0.8 ml
 +
| 1.2 ml
 +
| 1.6 ml
 +
|-
 +
| water
 +
| 1.4 ml
 +
| 2.8 ml
 +
| 4.2 ml
 +
| 5.6 ml
 +
|}
=====Solution III=====
=====Solution III=====

Revision as of 00:58, 26 February 2008

Back to Protocols

Contents

Plasmid DNA Miniprep

This procedure is used when many transformants need to be screened. The reagents are much less expensive than the minicolumns used for obtaining high quality pDNA. Typically once the correct transformant has been identified, a streak plate is prepared and then minicolumn pDNA extracted from an isolated colony (and a glycerol stock can be made).

Lysis by Alkali

Modified from Birnboim and Doly (1979) and Ish-Horowicz and Burke (1981)

  1. Transfer a single bacterial colony into 2 mL of LB containing the appropriate antibiotic in a loosely capped 15-mL tube. Incubate the culture overnight at 37C with vigorous shaking.
  2. Pour 1.5 mL of the culture into a microfuge tube. Centrifuge at (12,000g for 30 sec, or 5,000g for 5 min) at (4C or RT) to pellet the bacteria. (pellets can be frozen here)
  3. Remove the medium by decanting and inverting tube on paper towel, or by aspiration.
  1. Resuspend the pellet in 200 μl of ice-cold Solution I. (tubes can be stored cold for a few hours here)
  2. Add 400 μl of freshly prepared Solution II. Mix contents of the tube by inverting the tube rapidly five times. Store the tube on ice for 5 min.
  3. Add 300 μl of ice-cold Solution III. Vortex 10 sec. Store the tube on ice for 7-8 minutes.
  4. Centrifuge at 12,000g for 5 min at 4C. Transfer (~850 ul) supernatant to a fresh tube.
  5. Precipitate the DNA (add 500 ul isopropanol (tubes can be stored cold ON here); leave at RT 15 min; spin 5 min at 12K; drain; wash by centrifuging (12K for 5 min) with 500 ul 70% EtOH; drain; dry) and resuspend in 30 μl of TE (pH 8) (can be resuspended ON). TE may be prepared to contain RNase A at 20 ug/ml by adding 1 ul RNase stock (10mg/ml) to 500 ul TE. TE is 10 mM Tris pH 8.0 + 1 mM EDTA pH 8.0.
Solution I

50 mM glucose
25 mM Tris*Cl (pH 8.0)
10 mM EDTA (pH 8.0)

Solution II

0.2 N NaOH

1% SDS

Component < 5 samples < 10 samples < 15 samples < 20 samples
10% SDS 0.2 ml 0.4 ml 0.6 ml 0.8 ml
1N NaOH 0.4 ml 0.8 ml 1.2 ml 1.6 ml
water 1.4 ml 2.8 ml 4.2 ml 5.6 ml
Solution III

5 M potassium acetate 60 ml
glacial acetic acid 11.5 ml
H2O 28.5 ml

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