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(?)
Edited by student47, 24 September 2013 - 06:55 AM.