Hello, I am new to molecular cloning and I have been working on cloning my passage samples for almost a month without success. I was able to successfully clone my wildtype but it is incredibly challenging and frustrating for my passages. I am suspecting a low quality insert as the main root of the problem, but I maybe wrong. I wonder if anyone could help me understand what is causing my cloning to fail.
Basically, my insert (around 7-7.5 kb) is originally from the RNA I extracted from cell culture supernatant which I reverse transcribe then PCR. After amplification, I use approximately 200ng for infusion cloning. My vector is amplified from a different infectious clone which I double digest with DPN1 the gel purified. My insert shows relatively thin but bright bands on gel while my vector has thick and bright bands.
For infusion cloning I tried to use 1:1, and 1:3 insert-vector ratio and Takarabio infusion cloning enzyme (2uL). After overnight incubation, I get good colonies. The problem comes in after colony PCR. I amplify the front and back part of my construct to confirm successful ligation of my vector and insert however, I don't get the correct band sizes for most of the colonies. I should have 4kb for both front and back parts.
For infusion cloning I tried to use 1:1, and 1:3 insert-vector ratio and Takarabio infusion cloning enzyme (2uL). After overnight incubation, I get good colonies. The problem comes in after colony PCR. I amplify the front and back part of my construct to confirm successful ligation of my vector and insert however, I don't get the correct band sizes for most of the colonies. I should have 4kb for both front and back parts. I am thinking the bands appearing on my gel are unspecific amplifications which I am not really sure what caused it. I am attaching a photo of my gel for reference.
I tried re-extracting RNA from my remaining cell supernatants but I still get the same results. I am now at loss so any suggestions are highly appreciated.
I have faced this problem many times. When I work with clone screening, sometimes I get the right size bands in colony PCR and sometimes I don't get any. In the latter case, I generally try other alternatives for screening. I pick random 5-6 colonies and grow overnight in selection media (with antibiotic to ensure that only cells having the ligated vector will grow). Then next day isolation of plasmid (ligated vector) followed by quantification on nanodrop, electrophoresis on agarose gel. This gives me an idea that I have some ligated vectors present in my samples. After confirmation of the vector, I proceed with the PCR using primers on the flanking region of my insert. Here I usually get the right size band that I was not getting in colony PCR. If you want some more confirmation of insert ligation then you can try Sanger sequencing or restriction digestion with any enzymes whose site is present in your insert. I hope this will be helpful for you.
Dear Donna, sometimes when construct size is big you may get false results in colony PCR as mentined by Mohd Ahmad in such case you should follow another approach, If you have remaining white colonies you may go for colony inoculation in suitable media using correct antibiotic selection and go for plasmid isolation followed by restriction digestion. If you have vector map you can select R-sites that includes your insert too and go for digestion to confirm that your insert is there. If you do not get correct result eg 7 or 7.5 kb insert band means your product is not ligated. You can also go for another approach ie band comparison simple load your empty vector plasmid and your recombinant plasmid you will get a band shift as your rec plasmid containing 7 kb plus size will come above the empty v plasmid on gel.
From your gel pictures it is confirmed that you are taking too much cells from a single colony that may also cause amplification problem, so for safer side you pick a colony in PCR tube dissolve it in 50-100 ul ddMQ, put @94 degree C in thermal cycler (PCR machine) for 10 minutes to burst the cells after that give a short spin and than take 5-10 ul of that and add into the PCR reaction mixture. In this case your reaction mixture will be free from unwanted cell debris and other components. May be you get your clear result than. Hope this may help. Good Luck.
Thank you for your answers Mohd Ahmad and Shaifali Pal
I did colony inoculation then sequencing. However, insert sequence showed peaks that were either too noisy or none at all. The vector sequence on the other hand showed good peaks. I tried enzyme digestion as you have suggested. I used one cut enzyme (XhoI) and followed the manufacturer's protocol. After gel loading, I didn't get my expected band size, instead, I got bands of lower size and bright thick bands which seem to be uncut plasmids. Below, the left photo shows the uncut plasmid.
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