Hi all,
I'm trying to clone a shRNA in the pLKO-tet-on (21915, Addgene). So far, few clones have been obtained, but one issue is the quantity of plasmid i get from miniprep for screening. It is very low even with a lot of bacteria volume (I have tried 2, 5 and 10mL). So, it's hard to process for the restriction step coming after and select the positive clones.
For the few restrictions with XhoI I have done, I do not see the expected band around 200bp even if I digest the original plasmid.
Did you already have similar problems with this plasmid and could you provide me tips/protocols for bacteria culture or restriction reactions please?
Thanks a lot.
What bacteria line are you using to grow it up? Typically cells with a Tet resistance do not grow well. I usually need to use Top10 cells since typical DH5 alpha will not do (and even then I get very low transformation efficiency) : I will even go as far as to doing 2 dilutions and plating everything to get colonies. Did you try Terrific broth instead of a standard LB? I will do so for hard to grow plasmids.
I have tried LB. I used the strain stbl3 as recommended. Maybe i should grow more for preps. Was your cloning ok ?
Hello Cedric I hope you are doing well! Actually I just did this cloning and had the same problem as you. I get rid of it by using terrific broth instead of lb in stbl3 line. I never get big amount of dna though but sufficient to perform the cloning! Hope that help!
hi Rafik
Glad having news from you. I'm doing well except this cloning... Well I'll try terrific broth and let you know. Also did you have this weird problem with the Xho1 restriction?
For the original plasmid i think that you should have a band just above 100bp and a 100bp + 200bp for the cloned plasmid. The only issue i got with this is that because the fragments are so small, they didn't catch a lot of etBr and appear very faint. I attached a picture of one of my gel (11-4 is positive and 11-5 is negative).
Hi Rafik,
Indeed, Terrific seems to provide much more pDNA. just one last question, when you screen your preps, do you perform miniprep or directly midi preps? And what volume of bacteria do you grow for this? Thanks
Hi Cedric
I am glad it worked. For screening i did miniprep using 5 ml of TB. I then transformed a new batch of bacteria with my positive clones and proceed to maxiprep using 150ml of TB.
So I have successfully cloned shRNA into this plasmid. I used T4-PNK (NEB) alongwith 1ul of NEB ligase buffer to charge phosphate groups at the end of the oligos. This was done in PCR machine (37 deg for 45min and then 95deg for 5min then -1deg/20sec), 10ul rxn vol..
I know that double digest has the phosphate groups to work with but this has helped me a lot.
Then dilute the product to 30-50 times. Use 1ul to 2ul (You can use more but this T4 PNK charging will give you the result), 1ul (27ng) of double digested DNA and 1ul T4 DNA ligase (I used Takara but it hardly matters) and obviously buffer (Final volume of the reaction is 10ul). Then use 3 conditions (37deg, 4h; 16deg, 16h and 4deg, 48-72h). I have used DH5A and NEB Turbo (Ultracompetent cells), in NEB turbo you will get a ton of colonies but that will add up to your background as well. I got +ve colony from DH5A. I have screened 40 colonies and got positive in one of them. I had tried colony pcr but it gave me false positive. So RE digestion seemed to be more promising. Please note that I did 80 mini preps, without using kits, in 2 days and I was able to do that in 2ml MCTs with 1ml culture volume, you will get enough DNA to do screening (anyway you need 1-2ug for screening). I used 0.15ul of enzyme, XhoI, as it is pointless to spend much on enzyme if you want to see just 2 bands at 200bp. During minipreps I used RNase and PCI, but without these also you should get result.
DO NOT USE AMPICILLIN EITHER 50ug/ml (Too low as ampicillin will degrade over time and you will get stray colonies) or 100ug/ml(Too high, you will not get any colonies). USE CARBENICILLIN 50ug/ml (Final conc.) for making plates, it is a 4th generation antibiotics and lot more stable than ampicillin.
Hope this helps,
Happy cloning.
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