Plasmid mini-preps: purification of plasmids from E. coli

 

Using the Qiagen kit:

 

1. Add 2-4 ml of LB media + antibiotic to sterile, labeled tube.  Pick a labeled bacterial colony from plate using a sterile toothpick or gel tip and swish into the media to inoculate.  Grow the bacteria for 16-24 hours at 37 C and 200-225 rpm. 

1b) Pour bacterial culture into labeled 1.5 ml tube and spin cells down in microcentrifuge on highest setting for 1 minute.  Shake off media into flask containing soapy water.  Repeat this step in same tube to get bigger cell pellet. Cells can be frozen at this step and processed further later.

1c) Resuspend pelleted bacterial cells in 250 µl Buffer P1 – The bacteria should be

resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

Ensure that RNase A has been added to Buffer P1 – see check mark on top of bottle cap.

 

2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.

Mix gently by inverting the tube. Do not vortex, as this will result in shearing of

genomic DNA. If necessary, continue inverting the tube until the solution becomes

viscous and slightly clear. Do not allow the lysis reaction to proceed for more than

5 min.

 

3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube

4–6 times.

To avoid localized precipitation, mix the solution thoroughly, immediately after

addition of Buffer N3. The solution should become cloudy.

 

4. Centrifuge for 10 min at highest speed in a microcentrifuge.

A compact white pellet will form on the side of the tube wall.

 

5. Pour the supernatants from step 4 into labeled QIAprep spin columns.

 

6. Centrifuge for 30–60 s on high. Discard the flow-through – the DNA is bound to the silica gel in the column.

 

7. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for

30–60 s.

 

8. Discard the flow-through, and centrifuge for an additional 1 min to remove residual

wash buffer.

Important: Residual wash buffer will not be completely removed unless the

flow-through is discarded before this additional centrifugation. Residual ethanol

from Buffer PE may inhibit subsequent enzymatic reactions.

 

10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA,

add 40 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep

spin column (don’t touch gel matrix with tip), let stand for 1 min, and centrifuge for 1 min.  The flowthru contains your plasmid DNA.  Label tube with plasmid name, your initials, date.  Determine concentration using the NanoDrop machine.

 

 

 

Homemade plasmid mini-prep kit

 

Buffer P1

                        50 mM Tris HCl  pH 8.0

                        10 mM EDTA

(I use 2. 5 ml of 100X Tris-EDTA buffer from Sigma #T-9285 in 50 ml water)

                        100 ug.ml RNASE A

 

RNase A

Dissolve RNASE A powder in H2O to  give 100 mg/ml

Boil 5 min to kill DNase

Aliquot and store at –20 deg.

 

Buffer P2 (200mM NaOH, 1% SDS):

                        1M NaOH                               10 ml

                        10% SDS                                 5 ml

                        Water                                       35 ml

 

Buffer N3

                        3.0 M KOAc, pH 5.5 (29.5 g/ 100ml water, adjust pH with glacial acetic acid  )

 

Buffer PE 

75% EtOH.

25 mM NaCl,

5 mM Tris-Hcl, pH 7.5

 

Buffer EB

10 mM Tris·Cl, pH 8-8.5

 

Purchase Mini-spin columns from Epoch Biolabs, Inc., (www.epochbiolabs.com).  These columns are cheap and can be used for plasmid prep, pcr cleanup, gel extraction of DNA fragment from agarose gel, total DNA/RNA, genomic DNA, buffer exchange etc.

 

Protocol

Day 1: Pick a single colony from a selective plate and inoculate a culture of 2–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with vigorous shaking.

Growth for more than 16 h is not recommended since cells begin to lyse and plasmid yields may be reduced. Use a tube or flask with a volume of at least 4 times the volume of the culture. 

Day 2:

1. Pour culture into 1.5 ml microcentrifuge tube and spin 1 minute at 13,000 rpm in a table-top microcentrifuge. Pour off media and resuspend pelleted bacterial cells in 250 µl Buffer P1, using p1000 blue tip to resuspend cells. No cell clumps should be visible after resuspension of the pellet.

2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.

Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

 3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.

To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3.

 4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.

 5. Pour the supernatant from step 4 into a spin column.

 6. Centrifuge for 30–60 s. Discard the flow-through.

 7. Wash spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.

 8. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.

9. Place the spin column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 60 µl Buffer EB or water to the center of each spin column, let stand for 1 min, and centrifuge for 1 min.

10. Determine plasmid concentration using NanoDrop spectrophotometer.  Write DNA concentration on side of tube and store DNA at -20C.

 

Solutions for running Qiagen columns

1. You can buy these solutions, but it's much cheaper to make them yourself. The recipes that Qiagen recommends for these buffers have changed a few times in recent years. These recipes are from the Spring 1992 protocol.

(previous recipes tended to use buffers at pH's that were very far from their pKa's; the new recipes were probably intended to correct this).

2. It is important that these solutions be made accurately: if the NaCl concentration is off by a few percent, this could mess things up.

stock solutions:

0.5 M MOPS pH 7.0

209.27 g MOPS (use the free acid form that USB sells)

~750 ml dH20

pH to 7.0 with 10 N NaOH

make up to 2 liters with dH20

3M NaCl

350.6 g Nacl

make up to 2 liters with dH20

1 M Tris pH8.5

121.1 g Tris base

~750 ml dH20

pH to 8.5 with concentrated HCl (takes ~20 ml)

make up to 1 liter with dH20

store these solutions at room temp.

QC buffer

666 ml 3M NaCl

200 ml 0.5 M MOPS pH 7.0

300 ml EtOH

dH2O to 2 liters

check the pH and adjust to 7.0 (it should be close) with NaOH or HCl

QF buffer

833 ml 3M NaCl

100 ml 1M Tris pH 8.5

300 ml EtOH

dH2O to 2 liters

check the pH and adjust to 8.5 (it should be close) with NaOH or HCl

store QC and QF in tightly capped containers at room temp.

QBT buffer

500 ml 3M NaCl

200 ml 0.5 M MOPS pH 7.0

300 ml EtOH

3 ml Triton X-100

dH2O to 2 liters

P1 buffer

1M Tris HCl pH 8.0 2.5 ml

0.5 M EDTA pH 8.0 1 ml

10 mg/ml RNAseA, boiled 0.5 ml

dH2O 46 ml

50ml total

P2 buffer

2M NaOH 5 ml

20% SDS 2.5 ml

dH2O 42.5 ml

50 ml total

Apparently this solution can go bad due to oxidation: keep the container tightly capped.

P3 buffer

potassium acetate 25 g

glacial acetic acid 13 ml

H2O 87 ml

100 ml total

final pH should be ~5.5