Dear Prakriti

1. concentrations of 30-40ng/ul whilst low are not unexpected from mini preps

2. In general in my experience, I will tend to get back about 100ul of DNA @ 50-400ng/ul

3. That is total yields of between 5ug and 40ug of DNA

4. This is derived from a culture volume of 10mls

5. If you wish to increase your yield and concentration it is actually ok to grow larger overnight as the saturation capacity of most mini prep columns is about 100ug so you could use up to 30ml of over night culture

6. In fact if you know your plasmid is low copy number this is advisable

7. The other trick you can use for preps of low copy number plasmids is to grow the overnight culture (10mls to 30mls) in an enriched medium like 2 x YT or terrific broth instead of LB: This will result in higher OD 600 and thus more material to lyse to make DNA

8. For Sanger sequencing you require about 100ng of total plasmid DNA per kb of construct; Thus, for example if your plasmid plus insert is 8kb you would require 800ng of DNA: I therefore think that concentration of plasmid > 100ng/ul are ideal in terms of a manageable big dye volume sequencing reaction

9. Given that you are presently obtaining conc about half that I would advise prepping from 20ml overnights of either terrific broth or 2 x YT (in effect enriched LB)

Dear Prakriti

concentration of 30-40ng/ul are borderline ti obtain good dance e sequencing. Out actual provider suggest at least 50ng/ul but sometimes de obtain good sequences also with less amount but it depends Also from the quality of the dna.

i suggest to you ti try the ezna mini kit 2 of omega biotek thay starting from 10ml in my experiences with pet vector and cells as top10 it supply around to 200-300ng/ul

you can find more details about it on my blog:https://proteocool.blogspot.com

ProteoCool n°12

(E.Z.N.A HP MINI KIT II: MIDI KIT YIELDS from a MINI KIT) at page 6

good luck

Manuele

Dear Prakriti

1. concentrations of 30-40ng/ul whilst low are not unexpected from mini preps

2. In general in my experience, I will tend to get back about 100ul of DNA @ 50-400ng/ul

3. That is total yields of between 5ug and 40ug of DNA

4. This is derived from a culture volume of 10mls

5. If you wish to increase your yield and concentration it is actually ok to grow larger overnight as the saturation capacity of most mini prep columns is about 100ug so you could use up to 30ml of over night culture

6. In fact if you know your plasmid is low copy number this is advisable

7. The other trick you can use for preps of low copy number plasmids is to grow the overnight culture (10mls to 30mls) in an enriched medium like 2 x YT or terrific broth instead of LB: This will result in higher OD 600 and thus more material to lyse to make DNA

8. For Sanger sequencing you require about 100ng of total plasmid DNA per kb of construct; Thus, for example if your plasmid plus insert is 8kb you would require 800ng of DNA: I therefore think that concentration of plasmid > 100ng/ul are ideal in terms of a manageable big dye volume sequencing reaction

9. Given that you are presently obtaining conc about half that I would advise prepping from 20ml overnights of either terrific broth or 2 x YT (in effect enriched LB)

Dear Prakriti,

In this case, you may proceed to TA Cloning where you can clone in pTOPO vector. I have used invitrogen TA Cloning kit which worked for me. Good Luck.

Pratibha

Thanks for the suggestions Laurence Stuart Dawkins-Hall

Starting from 30 mL of LB media, I have been able to increase the plasmid concentrations to 70-100 ng/uL. However, I think the purity ratios were slightly compromised because the A260/280 ratios were off from 1.8.

I am still waiting for my sequencing results to come. Let's see how the results come look. Thanks again.