yes. please consult the Sambrook manual of Gene cloning (I), there are many protocols regarding plasmid isolation especially miniprep method of brimboin and dolly 1979 is quite easy in manual form.

We do Boil-prep (Holmes and Quigley, 1981) It has only the disadvantage that is not really clean, but if you scale it up, you can do phenol-chlorophorm extraction and you will have a nice and clean plasmid DNA...

Here it is... I hope it is understandable!

"Positive clones for selection were grown overnight on 1/6 of LB plates at 37°C, collected using a cotton swab and resuspended in 300 μl STET buffer. After adding 10 μl of a lysozyme solution, the samples were mixed gently by inversion and incubated in ice for no more than 5 minutes. To lysate the cells completely, cells were exposed one minute to 100°C. The cell debris, as well chromosomal DNA, was separated from the solution containing the plasmid DNA by centrifugation at 13000 rpm for 15 minutes. After removing the pellet with a sterile toothpick, 200 μl of isopropanol was added to the supernatant, mixed by inversion and incubated for 10 minutes at -20°C. The plasmid DNA was collected in a pellet after a 10-minute centrifugation at 13000 rpm. To eliminate any traces of isopropanol, awashind step with 500 μl of a 70% Ethanol solution followed. The solution was centrifuged at 13000 rpm for 1 minute and all the ethanol removed. After drying the DNA pellet, it was resuspended in distilled water or TE buffer.

STET: 8% Sucrose, 5% Triton X-100, 50 mM EDTA, 50 mM Tris HCl pH 8,0.

Lysozyme solution: 10 mg/ml lysozyme in STET buffer.

To purify and/or concetrate the DNA...

Plasmid DNA that was used for transfection experiments was concentrated and purified using phenol:chlorophorm. The solution containing plasmid DNA was mixed in a proportion 1:1 with phenol:chlorophorm solution (1:1) and vortexed (only for plasmids smaller than 10 kb). After centrifugation at 13000 rpm for 10 minutes at room temperature, the upper phase was collected and mixed with 3M sodium acetate (1/10 Volume). 9-Volumes Ethanol 100% were added and then mix by inversion. The resulting solution was centrifuged at 13000 rpm, 4°C for one minute and the supernatant discarded. The pellet was washed once with 500 μl ethanol 90%, centrifuged again at 13000 rpm for one minute, and the ethanol discarded. The DNA pellet was dried and resuspended in 50 μl TE buffer. The DNA concentration was estimated and the DNA solution was stored at -20°C until needed.

I hope this helps you,

ok,,i have found it

but may i ask you what the trouble in doing plasmid isolation and can you give me the trouble shoot of it ?

Nageena and Luisa, thank's a lot for your suggestion

you are always welcome

Plasmid Miniprep

1. Take asingle colony and added into LB media containing kanamycin 1.5 µL (stock solution must be from 50-30 µL and then incubate at 37oC overnight.

2. The following procedure is performed at room temperature.

3. Add 600 µl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube. The procedure may also be used with the classical centrifuge- based procedure for processing up to 5 ml of bacterial culture. The procedure should be modified as follows:

A) Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed.

B) Discard the supernatant.

C) Repeat steps 1A and 1B as needed.

D) Add 250 μl of TE or water to the bacterial cell pellet and resuspend completely.

For 10 ml of TE buffer

• 250 µL 1M Tris PH 7.5

• 250 µL 0.4 M EDTA PH 7.5

• 500 µL 20% (1M) glucose

• Final volume 10 ml by D H2O

4. Vortex and then ,add 250 µl of solution Buffer B

For 10 ml solution Buffer B

• 80 mg NaOH

• 1 ml 10% SDS

• Final volume 10 ml by D H2O

5. Mix by inverting the tube 4-6 times carefully and incubate for 5 min at room temperature

6. add 350 µl of solution Buffer C

For 200 ml solution Buffer C For 100 ml solution Buffer C

• 20 ml 5 M CH3COOK • 10 ml 5 M CH3COOK

• 108 ml 10% GuHCL (Guanidine chloride ) • 54 ml 10% GuHCL (Guanidine chloride ) (70.6996 gm )

• Final volume 200 ml by D H2O • Final volume 200 ml by D H2O

Adjust PH into 4 with imidazole

7. Mix by inverting the tube 4-6 times carefully.

8. Centrifuge at max speed for 10 minutes

9. Transfer the supernatant (~900 µl) into the column. Avoid disturbing the cell debris pellet.

10. Place the column into a Collection Tube and centrifuge for 1 min at max speed (DNA will be in the filter )

11. Discard the flow-through and place the column back into the same Collection Tube.

12. Wash the column with 500 µl of 1.5 M GuHCL (Guanidine chloride ) and Centrifuge for 1 min at max speed Discard the flow-through and place the column back into the same Collection Tube.

13. Wash DNA with 70% ethanol (500 µ l), and Centrifuge for 1 min at max speed.

14. Centrifuge for 1 min at max speed for the sample to remove residual of ethanol and buffers

15. Transfer the column into a clean 1.5 ml microcentrifuge tube then add 70 µl of Elution Buffer directly to the column matrix and let stand for 10 minute at room temperature.

1ml Elution Buffer

• 1 ml dD H2O

• 2 µl RNAse A

• 2 µl RNAse T1

16. Centrifuge for 1 minute at max speed to elute the plasmid DNA.

If you employ alkaline lysis such as described in Molecular Cloning or posted by Ahmed, the knacks are as follows;

1. cell resuspension completely before lysis: incomplete resuspension cause genome DNA contamination

2. immediate hardly mixing by shaking when you add lysis solution: slow and insufficient mixing cause genome DNA contamination

3. gentle mixing when you add neutralization solution: hard mixing cause impurity

Anyway, my easiest alkaline lysis protocol is as follows;

1. inoculate single colony in 1.5 ml LB with apt. antibiotics (depend on vector)

2. shake at 200 rpm, 37 degree C overnight

3. pour all culture into 1.5 ml micro tube

4. harvest cells by centrifuge at max speed (e.g. 12,000 rpm) for 30 sec

5. resuspend cells COMPLETELY in 200 ul TE buffer (10 mM Tris-HCl, pH 8.0/1 mM EDTA)

6. add 400 ul lysis solution (0.2 N NaOH/1% SDS, freash prepared) and IMMEDIATELY MIX HARDLY BY SHAKING but not voltex

7. leave on ice for 5 min (no more 5 min)

8. add 300 ul neutralization solution (3 M KOAc; mix 60 ml 5 M KOAc/11.5 ml acetic acid/28.5 ml water) and mix gently

9. leave on ice for 5 min

10. centrifuge at max speed for 10 min

11. remove supernatant to new tube with pipette

12. add 550 ul isopropanol and mix

13. leave for 20 min at room temp

14. centrifuge at max speed for 10 min

15. discard supernatant and add 1 ml 70% ethanol, then discard supernatant again

16. leave tube up-side-down on a paper towel for 5 min

17. add 50 ml TE buffer with 1 ug/ml DNase-free RNase A and dissolve the precipitate

18. leave for 30 min

19. add 5 ul of 3 M NaOAc, pH 5.2 and 125 ul ethanol

20. leave for 20 min

21. centrifuge at max speed for 10 min

22. discard supernatant and add 1 ml 70% ethanol, then discard supernatant again

23. leave tube up-side-down on a paper towel for 5 min

24. add 50 ml TE buffer

You can use the plasmid for restriction digestion, transformation, sequencing etc. If you need more pure one, you can extract with phenol/chloroform to remove protein.

I hope it will be helpful for you.

all: thank's so much