There is a protocol, seems a bit unorthodox but it worked fine for me.

Here's what I did:

First I run the gel (obviously)

Then I sliced the band corresponding to the DNA I wanted to purify (in my case it was the section of the gel containing fragmens from 1000 to 3000 bp, so It should not be a problem for your 11 kb plasmid).

You need to have a pipette tip like the one in fig 1. You have to cut the pipette tip at the mark shown in fig 2.

Then you put your sliced pipette tip into a microfuge tube. Leave it somehow loose, if you press it then it might get stuck.

Then put the gel slice into the pipette tip, the filter prevents it from falling to the bottom of the tube, and it will retain all the agarose.

Transfer your tube to the ultrafreezer, and leave it there for 20 minutes.

Transfer your tube to the microfuge immediately after the 20 minutes and spin it for 10 minutes at top speed.

The agarose will not pass through the pores in the filter, but the DNA will, so, after the centrifugation step you'll have the agarose trapped in the filter, your DNA will be at the bottom of the tube along with the TBE or TAE buffer, it's just a matter of removing the sliced pipette tip and then you can purify your DNA with a simple extraction (phenol, phenol/chloroform or just chloroform), followed by EtOH precipitation.

I know It sounds quite unorthodox, but it works, I constructed a genome library using purified DNA with the method above, and in the first trial I obtained ~600 clones per transformation cuvette.

There is a protocol, seems a bit unorthodox but it worked fine for me.

Here's what I did:

First I run the gel (obviously)

Then I sliced the band corresponding to the DNA I wanted to purify (in my case it was the section of the gel containing fragmens from 1000 to 3000 bp, so It should not be a problem for your 11 kb plasmid).

You need to have a pipette tip like the one in fig 1. You have to cut the pipette tip at the mark shown in fig 2.

Then you put your sliced pipette tip into a microfuge tube. Leave it somehow loose, if you press it then it might get stuck.

Then put the gel slice into the pipette tip, the filter prevents it from falling to the bottom of the tube, and it will retain all the agarose.

Transfer your tube to the ultrafreezer, and leave it there for 20 minutes.

Transfer your tube to the microfuge immediately after the 20 minutes and spin it for 10 minutes at top speed.

The agarose will not pass through the pores in the filter, but the DNA will, so, after the centrifugation step you'll have the agarose trapped in the filter, your DNA will be at the bottom of the tube along with the TBE or TAE buffer, it's just a matter of removing the sliced pipette tip and then you can purify your DNA with a simple extraction (phenol, phenol/chloroform or just chloroform), followed by EtOH precipitation.

I know It sounds quite unorthodox, but it works, I constructed a genome library using purified DNA with the method above, and in the first trial I obtained ~600 clones per transformation cuvette.

Fig_2

You may use Slica Beads DNA Extraction Kit from Thermo Scientific (http://www.fermentas.com/en/products/all/nucleic-acid-purification/kits/k0513-silica-bead-dna-extraction) or use Wizerd SV Gel and PCR Clean Up system from Promega (http://www.promega.com/products/dna-and-rna-purification/dna-fragment-purification/wizard-sv-gel-and-pcr-clean_up-system/). I have isolated similar sized DNA fragments with very less loss from Slica beads kit and a bit smaller like 8 kb from Wizer Gel and PCR clean up kit, both worked very well! Best of luck!

Hmm innovative technique boss @victor flores...good

i think instead of pipette tip we can use catridge column which we use for RNA extraction:)

If you know someone with a Sage Science Blue pippen prep you can try it. You load your sample into a pre-cast gel cassette and simply type in the size range you want to extract and the machine does the rest. Their 0.75% gels say they work up to 10kb but I bet you can go one kb higher.

http://www.sagescience.com/bluepippin/

We use the protocol from http://kirschner.med.harvard.edu/files/protocols/geneclean.pdf

14 mL of NEW concentrate + 280 mL of DI H2O, mix

Add 310 mL 100 % EtOH, mix

Store NEW Wash at -20C

1. Add 3 volumes of NaI stock to band (~250 + 750 uL NaI). Incubate for 10 min. at 45 –

55C to melt agarose.

2. Vortex Glassmilk mix until there is an even suspension. May take a minute or so –

hold tube horizontally. 5 uL of Glassmilk to 5 ug of DNA.

3. Add 5 uL of Glassmilk to DNA / NaI solution. Mix and place on ice for 15 min. Mix

every 1 – 2 min. to keep Glassmilk in suspension. If volume is > 1.5 mL allow 15 min.

binding time.

4. Pellet reaction – quick spin. Remove supernatant. Respin. Remove remaining

supernatant. Wash pellet 1 X with NaI stock (200 – 400 uL). Spin. Remove

supernatant.

5. Wash pellet 2 X with NEW Wash:

Add 10 – 50 volumes of NEW Wash (200 – 700 uL). Resuspend with pipet tip. Spin.

Remover supernatant. Repeat 2 X.

6. Elute DNA from glassmilk beads.

Resuspend pellet in TE buffer (amount = amount (uL) of Glassmilk added in step 3.

Incubate at 45 – 55C for 2 – 3 min. Spin 30 seconds. Remover supernatant + DNA to

fresh tube. Repeat elution 1 X.

7. Spin supernatants 1 X again. Remove to fresh tube. 10 uL 􀃆 2 ug.

Alkaline lysis is the best method to purify any plasmid upto 20 kb. You can also try the Qiagen plasmid extraction kit to get pure plasmid

Sigma gel extraction kit is best for DNA extraction and purification from gels. use 0.7% agarose gel to run your products. one purification can easly give you 5-6µg pure product.

Conventionally: Glass wool (silane treated), tubes and needle. Commercially: QIAEX II Gel Extraction Kit

Dear Lakshmi Narasimha, I used catridge column from a RNA extraction kit for Plasmid extraction, lower extraction

Qiagen gel extraction kit works well

I use GenElute Five-Minute Plasmid Miniprep Kit from Sigma-Aldrich and a similar kit from qiagen. All perform well with this plasmid size. Your problem will be the amount/yield. You shold look for the diffrent size of colum they have in catalogs. probably one fit with your needs.

you can easily use qiagen gel extraction kit but remember to incubate the spin column at 37C for 2 mins after adding elution buffer.

Hi Aditi,

I used to isolate 23 kb plasmids using Qiagen's mini plasmid purification kit. Use of pure water and reloading the eluate multiple times to elute the DNA may help. For 80 to 100 ug plasmid use the appropriate size. For gel extraction, as Alicia Sanchez mentioned above, low melt agarose should work. Alternatively, use Qiagen's plasmid purification buffer set (without the columns) and precipitate the plasmid using isopropanol. The prep would be relatively pure with little or no RNA or genomic DNA contamination.

Hi Aditi

Most column based protocols work very nice till 10kb range of fragments..after that elution efficiency can decrease significantly......You can also try Low melting point agarose based separation, followed by 'Agarase (NEB)' based degradation of agarose slice and recovery of plasmid by regular plasmid purification protocols or that given by the NEB.....this also works fine

bye

Aditi, Which system did you use finally? I need  to do something similar to your question, and would like to learn from your experience. Thank you.