My vector in which I cloned shRNA primers have a very high copy. I cloned shRNA plasmids and then tried to transform them in Stbl4 but I'm getting very very low or almost negligible concentration of DNA by both Mini prep & MEdi prep. I need DNA to proceed with lentiviral production. What could be the reason for this problem? Bacteria are growing fine in LB broth and it turns turbid too.
are you using antibiotic?
once I had this very same problem, it turn out that the ampicilin I was using was not working, leading to overgrow of non transformed bacterias or loss of the plasmid
Thankyou Beatriz , but I've already checked my antibiotic, thats fine and I dint had this problem with any other transformation except ones in Stbl4
I never used Stbl4, my suggestions are:
1. Stbl3 works really well, we can get 03-0.6 ug/ul pLV plasmid DNA in 100-120 ul eluent from 5-7 ml LB overnight culture per miniprep. But please note Stbl3 is endA+
2. You may want to inoculate colonies in 5-7 ml LB + antibiotic in the 15 ml tube and shake at 300rpm for 20hr, it may help.
Best Wishes.
Low yields for shRNA plasmids is not unusual. Try using a 2ml terrific broth culture grown for 16 hours.
@Wen-Yuan Hu -Thankyou, I've ordered Stbl3 but I've to repeat all steps again so to save time i was wondering if i can continue somehow with Stbl4
@John : Thankyou , I know they will give less yield in comparison but problem is its almost negligible DNA yield . I will try with Terrific broth.
For my shRNAs I have always used XL1-Blue Competent Cells
(Agilent, Catalog #200249). Always with very good results
Disha, you can inoculate Stbl4 in 5-7 ml of LB and shake @ 300rpm for 20hr, then spin down the bacteria pellete with desktop centrifuge such as Sorvall RT6000D, add solution 1,2,3 to start the miniprep.
Dear all Thanks for your previous suggestions I tried Transforming my ligations with Stbl3 , they grew very well just like stbl4 but when i did miniprep though the yield is bit more than what i was having with stbl4 but still 1/3rd of what we usually get like now for one such clone i'm having 122ng/ul...what can i do to have good DNA yield???
Did you finally solve your problem, Disha? I am facing similar problems when extracting a 20 kb vector from Stbl4 ...
I have Sure, Sure2, Stbl3, and GT116 cells on hand for inverted repeat cloning. I prefer the latter two, but sometimes the other 2 work better. Its a hit and miss with these. The 122ng/ul is not that bad. Anyways, you can try 2xLB which has all the ingredients at double the amount except for the salt. That increases the yield too, and is easier to make than the TB.
@Mikel Irastortza: Nope I could not solve the Problem using different media too so finally i retransformed them into commercial Stbl3 and with that it worked wonderfully.
@Koen Venken : Thankyou for your reply, I knew Stbl3 will work but i transformed them in Stbl4 as that also should have worked but it din't and now I'm back to Stbl3
@Mikel Irastortza: I read about Stbl4 and found out that for type of work I was doing it wasnt very well suited choice may be you should also check and change it
@ Disha Chauhan: I changed some growth conditions and eventually solved my problems with this vector, thanks anyway!
Hi disha,
I face the problem of low yeold as well. I am using psuper retro vector for cloning my shrna. However i get low yeild of plasmid after isolation. Say for example from 4 ml cuture total plasmid i get is hardly 2-3ug. The oligoengine protocol which supplies this plasmid suggests using dh5alpha for growing plasmids. I tried lowering salt concentration in or der to improve yeild. Can you tell me what are the growth conditions you used for culturing bacteria for plasmid isolation?
Thank you
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