Wise expertise needed......Puzzled by something minor..concentration of annealed - (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!
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!
OK, I know what you mean by the confusion of listening to three differentpeople give you four opinions...
1. Annealing. I used to always anneal in TE (because it used to 'always' work) until 1 month ago. I now anneal in NEBuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9 @ 25°C). I mix the oligos, make it 1x buffer, boil up some water in the microwave, and place the tubes (in a floatie) into the water, then just leave it to cool on the bench. Works a treat.
2. Digestion. If the buffers are compatible, you should be able to do it. If, however, you suspect you're not getting 100% cutting, do sequential cutting, adjusting as needed. Start with the low-salt buffer, then add NaCl to get to the higher salt conditions.
3. Gels. the postdoc is right; for a 6kb plasmid, use 0.8% gel. When you dod the gel, run some undigested vector, and some single-cut vector so you get used to the different way in which DNA runs as supercoiled, single cut and nicked DNA.
4. Post-digestion treatment. If you're not confident you have completely-digested vector, treat with CI(A)P (calf intestinal (alkaline) phosphatase). Very important point: use only as much CIP as the protocol tells you, which means you need to calculate how many pmol of vector you have. If you heat-kill the CIP, an excess of enzyme can cause the DNA to precipitate!! Equally important: if you do CIp-treat the vector, you must treat any PCR product with a kinase, or there won't be any phosphates left for the ligation step.
Thanx Swanny!
Update:
1. I did a sequential digest yesterday, checking material after single digest, adding single-cut and uncut controls on the final gel. I then did a test ligation, purified double-digested vector plus ligase. I wanted to see if the overhangs were ok so I thought about ligating the stuffer sequence that I had just cut out back in again.......unfortunately I got TWO stuffer bands on the gel lol
When I did a double digest I only got on band at around 1000 and now I have two, at around 1000 and 900, hehe cloning doesnt stop surprising me
2. Ill check my annealed oligos on a 3%gel should be able to see difference right? Ill also do a test annealing in NEBbuffer2 and run that alongside the others see what I get.
I believe the end concentretion of your duplex is 2.5ug/ul rather than 0.25ug/ul
and I believe that your annealing is ok . you can use TNE buffer or even RNAase-free duplex buffer designed for dsRNA annealing
Update:
1. I did a sequential digest yesterday, checking material after single digest, adding single-cut and uncut controls on the final gel. I then did a test ligation, purified double-digested vector plus ligase. I wanted to see if the overhangs were ok so I thought about ligating the stuffer sequence that I had just cut out back in again.......unfortunately I got TWO stuffer bands on the gel lol
When I did a double digest I only got on band at around 1000 and now I have two, at around 1000 and 900, hehe cloning doesnt stop surprising me
2. Ill check my annealed oligos on a 3%gel should be able to see difference right? Ill also do a test annealing in NEBbuffer2 and run that alongside the others see what I get.
When you do your gels, could you put a copy on your next posting, so we can see what you're getting?