No insert for PCR cloning and restriction enzyme digestion - (Sep/24/2013 )

HI all,

I am not getting any inserts on my experiments.

it is a primer designed with xho1 and sal1 and vector is pmirglo. the xho1 used is from roche at .5 microl for 1 hr  and sal1 is from new england biolabs at .4 microl.

after intial pcr where i got the gene, i cut with xho1(.5 microl) and cleaned it and then (.4 microl) sal 1 (paralelly with vector was digested too) and cleaned up using qiaquick purification kit. than ligated at 3:1 ratio of insert:vector with 1 hr ligation at 26 deg c and .2 t4 ligase. thentransformed it to dh5e alpha competent cells via heat shock and seeded on ampicillin mixed plates. i have around 10 colonies each and grew it on suspension overnight and extracted plasmid via midiprep, then cut eh plasmid with xho1 and sal1 sequentially. at the end of second digestion it was not cleaned and directly added the loading dye to run on gel.

the gel is attached, lanes in order from left to right are - ladder (1kb), then genes in all lanes.

the problem is that the vector should be around 7 kb while here it stands above 10 kb. this was later rectified, when i ran the gel longer, some faint bands came to 7 kb ladder correspondence. since it was faint am guessing some digestion is not working.  genes am looking for are 500bp , which around the lowest band of ladder here.

my guesses are :

most strongly i think - one or both the enzyme is not working or buffer or ligation enzymes?!

something wrong with the competent cells/ligation?

am thnking of running a gel after each step to check if the digest worked or not since the linear plasmid cut with even a single enzyme should run faster. (?) also if i run the vector after ligation, should run slower than unligated one(?)

please help.

OK, sounds like you need to test the digests first - single cut plasmids should run at the correct size for the linear fragment; intact can run in a number of sizes, depending on the coiling state.  Make sure you use the correct percentage gel to resolve it properly.

As for the digests you haven't actually told us what you have done, 0.5 ul doesn't mean anything - what reaction volume?, how much DNA?, how many units of RE did you use? Are you sure that you are digesting the right DNA?  How was the DNA prepared?

Ligations: was the 3;1 ratio a molar ratio? did you try any others?  did you try longer ligation times and different temperatures?

Could your primers be wrong?  Did you add some bases 5' of the cut site so as to give the RE something to bind to?  Did you get the cut site correct?

hi bob1,

thanks for your advice, i'll explain the empty details i left too. i have checked the plamids and digest and just found out that one of the enzymes of the double digestion, was not working well. I am going to try if a higher concentration will work. it was resolved on 1 percent gel. ,5 ul was the volume of enzyme used, with 6 ul of gene product with 40 ng/ul concentration, extracted from gel after pcr. enzymes used were around 1 unit per reaction of total 10 ul volume. tthe pcr product ran on the right size on ladder, so i assume it was the right size. i did try other ratio's of ligation which did not work. i do have 4 bp ahead of the restriction site. the cut site is correct as per multiple sources.

I am going to check that if only one restriction enzyme was working, could i still have the insert?!. apart from that am still waiting for new enzymes to come.

This is likely a classic case of inhibition due to having too much of the volume of your reaction from "purified" DNA. Six out of ten microliters of your reaction come from the DNA. Small amounts of inhibitor in the isolated DNA can prevent reaction. These can be ethanol (most likely) or Gu-HCl from your gel purification reaction. You want to do this reaction in at least 50 ul final volume. Retry the reaction with

6 ul DNA

5 ul  RE buffer (10x)

1 ul RE

38 ul water

Tehre is a reason the protocols for your restriction enzymes do things in higher volume. If you want a smaller volume reaction, you often need to cut less (volume of) DNA.

i will try that .. thanks..