earlier when i used this kit some 15 days ago i got great results but now when i am trying for the same.. my results r going down. dont know the reason behind. conc. of cells.. there phase growth everything have been checked. yet cant find the error
I'd recommend to grow a fresh broth of your transformed cells and try isolating plasmid while the cells are in viable growth phase (16hours).
Initially take atleast 5 ml broth for each extraction. I.e after u centrifuge 1ml, discard supernatent. Add another 1ml, spin, discard. This will help u in higher plasmid concentration.
Also at the end resuspend your isolated plasmid dna in less volume I.e if 60ul is recommended, you can add 30ul instead.
I hope this helps.
- Nice answer from Khudeja
- I would add to that by asking how old are your transformant colonies? More than 1 month?
- If so they can dessicate and the viability becomes lower resulting in poor yield
- If so retransform; Pick a colony and grow during the day @ 37C (starter culture)
- Then add 1/1000 aliquot to your overnight culture and grow for no more than 18 hours to ensure, as Khudeja has stated that they are till in (but near the top) of exponential phase and thus fully viable
- In addition, you could increase your growth by growing in 2 x YT instead of LB; In effect a richer version of L (google formula)
- You can also increase recovery from your column by adding water or TE (or what ever your elution buffer is) pre heated to 37C (instead of straight out of the bottle @ room temp); add to the column; wait 5 min and then spin through: You could do this x 2 with say a 30ul aliquot of pre heated elutant and then a 20ul aliquot of elutant; each time waiting 5 min having added to the column before spinning to recover your DNA
Laurence Stuart Dawkins-Hall Thank you so much for the recommendation. Recommendations like these by such great scientists keeps us motivated.
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