problems with recovery of dna from gel and ligation - (Sep/12/2012 )
1. I am digesting pNL4.3 (2 ug) with NheI and Sal I. After digestion I need both fragments generated by digestion, Bigger fragment is 13kb whereas the smaller fragment is 1 kb, I am using Nucleospin Gel extraction kit to purify from the gel (as this kit can purify dna fragments greater than 10kb),
After extraction from the gel, I quantified with Nanodrop, I got only 1-2 ng/ul of both fragments, Is it normal to get such less amount of DNA after recovering from the gel ?
2. Secondly I made the ligation with 25ng vector (pNL4.3 without Sali-NheI fragment) and insert (SalI-NheI fragment) from other plasmid, and used different ratios of vector:insert like 1:2, 1;3.. Only 1;2 reaction worked but I got very few colonies gathered in the center of the plate. when I miniprep these colonies I can recover only 3-5 ng/ul of this plasmid (ligated plasmid). what is the problem here ?
3. Is the restriction enzymes digested DNA (open dna) is stable inside bacteria after transformation and bacteria can grow normally ?
1) Gel extraction typically gives poor yield. Try TAE instead of TBE, as it works better for gel extraction.
2)Were our ratios molar ratios - not amount ratios? Low amount from miniprep - you should check that it is actually plasmid - run some on a gel. It could be that the plasmid is toxic.
3)The linear DNA tends to get degraded.
1) I am using TAE Buffer but still having bad yield.
2) I calculated the molar ratios from website that contains ligation calculator.
I checked it by running on the gel and it shows a DNA band above the unligated vector.
Im having Problems, extracting my DNA as well with the QIAGen extraction Kit. I follow the protocol as mentioned but when I elute the DNA in sterile water (20ul). The consistency of the extraction is like a gel. Im not sure If Im adding too much Mixture into 1 column or Something is wrong with the gel extraction. If there is something you guys suggest to me I would appreciate it.
Thanks Att; E
Wash the column with extra buffer QG before the PE wash. Wash twice with PE and elute in TE (rather than water).
shoaibarif: Colleague used Qiagen QIAEX II Gel Extraction Kit for larger inserts (up to 50 kb), and MinElute Gel Extraction kit for smaller ones (up to 4 kb, lower elution volume).
However he was saing that handling the QIAEX wasn't much comfortable, because it's some silica particles you need to spin down all the time and not a column.