Plz help me troubleshoot subcloning! - (Dec/22/2005 )
My steps are
1 sample (NR1, 7.2kb, 450ng) is double digested by HindIII (1ul) & NotI (4ul) (Buffer R), while 3 samples (NR2B, 8.5kb, 800ng, NR2C, 8kb, 540ng & NR2D, 9kb, 520ng) are double digested by EcoRI (1ul) & NotI (1ul) (Buffer O). Another sample (NR2A, 11kb, 800ng) is single digested by EcoRI (1ul) (Buffer EcoRI).
* The vector used is pBluescript SK- (2961bp). Total vol is 20ul. Incubation: 37C, 2hrs.
2. Vector (pcDNA3.1-, 5.4kb)
1 sample (V1, 2ug) is double digested by HindIII (1ul) & NotI (4ul) (Buffer R), while 1 sample (V2B, 6ug) are double digested by EcoRI (3ul) & NotI (3ul) (Buffer O). Another sample (V2A, 2ug) is single digested by EcoRI (1ul) (Buffer EcoRI).
* Total vol is 20ul. Incubation: 37C, 2hrs.
II. Gel Electrophoresis
1. 120V, 45min
2. stain w EB for 15min
3. cut bands under UV. Sizes are:1, 4.3kb; 2A, 8.1kb; 2B, 5.6kb; 2C, 5.1kb; 2D, 6.1kb.
III. DNA Purification (use Perfectprep Gel Cleanup from Eppendorf)
previously digested vector (pcDNA3.1-): 50ng; T4 DNA ligase: 1ul
* I made molar ratio of insert/vector be 3&1.
V. Transformation by electroporation
1. Take 2ul of each ligation products to 100ul cells. (1 negative control: 100ul untransformed cells were put into 1ml SOC.)
2. After electroporation, cells in 1ml SOC are incubated at 37C, 2hrs w shaking.
*self-prepared Dh5alpha electrocompetent cells are used.
VI. Transformed Cell Plating
1. Undiluted: 200ul
2. 30X dilution in LB medium: 100ul
3. overnite incubation at 37C
* plates used: LB w Amp. duplicates were done. for negative control, 200ul were plated.
The prob is I saw no colonies growing on the plates. I even incubated for another 24hrs, but still got no colony. so can anyone help me troubleshoot my steps? thnx a lot!
there are many places that could be the source of the problem. the first thing I would suggest is adding a positive control, and also plating your 'transformation control' on plain LB to make sure your media is OK and your cells are viable.
once these 'basics' are covered, if you still can't find the source of the problem, what I would do is go to google, type in "cloning troubleshooting". I did this and I was going to send you a link, but a ton of good stuff came up. You can go to many websites, Promega, Invitrogen, etc etc, there are even some links to this website and that search is probably the easiest way to get some troubleshooting tips
oh, hey, yeah, and why are you ever using more than 1 ul enzyme in a 20ul reaction? if it is a poor reaction, more enzyme will likely make the problem worse, not better
hi, aimikins, thnx a lot 4 ur advices! e reason why i use 4ul for NotI is bcoz it doesn't have high activity in buffer R. Wat is suggested in fermentas double digestion website is to use 4-fold enzyme.
I repeated digestion yesterday nd found out even my digestion is incomplete, so somebody suggested me to incubate overnite instead of 2hrs.
With 4 ul in a 20 ul reaction, you have far too much glycerol (10%) for most restriction digests. If you want that much enzyme, you should be using a much larger volume of water/buffer.
ic ic. this time i changed e total rxn vol to 40ul while enzymes remained unchanged.
I repeated digestion yesterday (increasing plasmid amount to 2-3mg nd changing total vol to 40ul), but after gel electrophoresis, some samples appear undigested (a band near to the well). Therefore, I repeated today nd decided to incubate overnite. Hopefully can observe desired bands 2mol.
A band near the well is not undigested plasmid, it is genomic DNA. It sounds as if your plasmid prep is questionable. How was it done? I assume you really meant 2-3 micrograms, not milligrams. I would be doing these is 100 ul reactions. You could try cutting with each enzyme separately to verify that each was really cutting.
You say that your enzymes are not completely cutting -- how do you know this? If it is just genomic DNA, then it may not matter (but you probably want to do the prep again to get rid of it).