Hello everyone,
We are trying to get some constructs after ligation of the plasmid and the insert of interest, and our efficiency rate is very low. Out of 10 colonies, usually only 1 or 2 (or none) is positive, since we are only working with the ampiciline resistence that the plasmid contains, therefore we have no way of knowing which colonie has the insert, it´s not like the whole X-Gal system.
In order to save some money, we are trying manual minipreps instead of kit minipreps in order to perform the later restriction analysis or even sequencing since it´s quite a waste of money to perform 20 miniprep kits to get a total number of 0 positive colonies, or even just one or two.
However, they look like this. I can never see a clear band like with the miniprep kit. Any ideas? what are we doing wrong?
These are the buffers I´m using:
P1 Resuspension buffer (100ml) --> 0.6g Tris Base, 0.37g EDTA, pH 8
P2 lysis buffer (100ml) --> 2 ml NaOH 10M, 10 ml SDS 10%, 88 ml H2Od
P3 (100ml) --> 60 ml Potassium acetate solution, 5M, 11.5 ml Acetic acid, 28.5 ml H2Od
The protocol is a normal one, I believe, resuspend pellet in P1, then add P2 and P3 after the adequate mixing, centrifuge, mix supernatant with 0.7 vols of isopropanol, centrifuge, clean pellet with cold EtOH 70%, let dry and resuspend in 50 ul of H20mq.
Any ideas? It´s just a waste of time and I always end up using the miniprep kit...
Thank you very much
Bea
Beatriz María Fernández Santos
It looks like from the image and your protocol that you are loading circular plasmid DNA, and not linearizing it. For checking on a gel you want to conduct a restriction enzyme disgest (on a small aliquot since you wont be able to recover it) and then load the linearized DNA onto a gel
Hi Bea,
When you do a manual purification of plasmid (instead of kit), you always get a smear like this. this DNA is not good for RE and cloning.
If you are getting 1 positive colonies out of 10, then your RE digestion is not 100%. Give longer incubation times for your vector and inserts with respective Restriction enzymes. when you have your vector and insert 100% cleaved on both ends, you will have 100 % success in colony screening.
But, before digesting the vector with REs, don't use the vector purified from manual method, use only kit purified one.
good luck.
Bhanu
Hello everyone,
I just wanted to let you know that apparently Shawn Krueger was right :) because I tried performing a restriction analysis as I would do with my miniprep kit in order to see if digested gel would look better and it looked perfect (+ the dirt fog below, of course, but that doesn´t motter) and even allowed me to see which colony was positive or had not been digested.
And Bhanu P Jagilinki, thanks for your answer. We however make sure the insert is 100% digested before proceeding to cloning by performing a insert-insert ligation, in which case, if you are able to see several bands multiples of the size of your insert, it means it´s digested in both sides because it was able to join itself several times. And all of my inserts have done that. So I truly don´t know why our efficiency rate is so low, we perform the ligation in a 1:3 plasmid:insert proportion.
Thanks again
Bea
Beatriz María Fernández Santos
Im glad it worked out for you. Also you can probably reduce the amount digested, based on the image it looks like you can get away with alot less input to visualize the screening.
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