Please answer these questions for me ! - (Sep/03/2008 )

So today, after my cloning didnt work yet again, I found out from a postdoct that a whole bunch of things were wrong with my cloning strategy. It was all very amusing, I seem to have to supervisor so I ask here and there, mainly another PhD student thats leaving the lab soon unfortunately. It seems that my Xho1/Bgl 2 double digest hadnt worked. I had a whole lawn of bacteria on my agar plates this morning and since this is most likely due to religation. I checked my control gel and saw that due to the size of my vector (6.4kb) and the gel percentage (1%) I probably hadnt separated the supercolied and cut vector bands properly and they had appeared as one. Since Im not experienced in this field I just saw the stuffer sequence and was happy that it had been cut.

Then the PhD informs me that shes had trouble with the Promega enzyme before maybe I should use another one.

Then a postdoc comes up to me and says she would have used a 0.8% gel and she wouldnt have done a double digest but a sequential one and that she had a buffer that worked for all enzymes called RB 100, that she wouldnt cut 10migrog in one go but 5 in 100microl.
Then she mentioned that the concentration i had written down for my annealing wasnt right

I had used 10 microg primer fw and 10 microg primer rev in 40 microl endvolume

She says endconcentration is 0.25 microg/ microl

PhD told me 0.5 microg/microl and thats the concentration ive been using



Whats the deal???


This is really strange, I run around like a chicken with my head cut off asking here and there, im learning a lot but still....this is a bit frustrating, everyone is going on holiday in september and october, my supervisor , the PhD, the technical assistants.

Im used to working independently but I should know small things like what enzymes work and that the thermocycler isnt good for annealing, that a waterbath should be better.

I wanted to be sure that the annealing had worked but they all say that annealing always works, always in water or TE.

Ive been reading this forum and it doesnt seem to be true.

Ok enough venting I hope you can give me some advice, thank you!

Hi,

Well you have 2 things which are the problem.

First, the digestion of the plasmids with XhoI and Bgl2. We normally use NEB enzymes so I cannot comment on other brands. You could do a double digest in NEB buffer3 for XhoI/Bgl2.

you should have runt he digested product in a 0.7-0.8% gel and make sure you run it for a long time to separate the cut from supercoiled DNA. You can digest 5ug of DNA or even 10ug but you need to have atleast 50ul reaction. You could definitely go up on the volume and also use a little bit more enzyme.

Annealing is a different thing. if there are discrepancies with the concentrations of the oligos, go to the stock solution and start from there.
We have a buffer which we use for annealing. But different labs have different protocols for annealing.

Knowledge is gained gradually and as long as one is in quest for knowledge, it will surely come.

Good Luck !!!

Thanx!

Do you think, if I having trouble digesting with Promega, Roche, Fermentas enzymes although im using a lot of enzyme maybe theres something wrong with my Maxi.
When I received it I asked if i could sequence it and I was told it should be ok and that was that.

Or maybe enzyme overload affects the reaction?

I remember when I cloned before there was this math formula used to figure out how much enzyme to use using a reference genome from the NEB catalog and whatnot. I only used NEB enzymes then and I did 4 constructs in a couple of weeks real easy.
If I start of digesting 2 µg of vector, how much enzyme should I really use? My boss said a 4x overdigest, so 8 units. Thats why I used 20 units for 5 µg but I think other people use waaaay less enzyme right?


I actually like learning about this a lot, I love this forum! And im gradually getting a clear picture of things and deciding for myself. For example I was told to calculate my vector concentration using a gel, comparing marker bands and such because the photometer was inaccurate. To be entirely honest ive always used the photometer for everything and i always got results. We also have a nanodrop that I could use. What do you think? Should I use the gel even though I have a tough time estimating whether im seeing 40 or 50ng of vector compared to the marker band?

Enzyme overload is bad because too much enzyme will end up chewing the properly cut ends and destroy the sites eventually. Each enzyme is different and for some enzymes I am very careful as to how much units I use. You could say I usually use 2units per ug , this is just a hint not something I stick with. You will learn as you gain more experience.

Regarding concentrations, I usually somehow feel the photomoter's are not very accurate. For nearly all small amounts of DNA, I prefer comparing marker bands.

It is difficult to say if a band is 40 or 50 ngs. Its just an approximate value. Initially, i was struggling with the same thing, but you will as mentioned above gain this with practice. As long as you donot mistake between 50 and 100ngs, its fine.

Don't worry too much and have a good weekend.

Cheers