Ligation - (Sep/07/2012 )

ascacioc on Sun Sep 9 10:36:56 2012 said:

Do not worry You are so panicky Relax, we will make this thing work... or at least try hard.

With what your supervisor says: it is not very wrong what she says; I mean it works like her as well, but it is better to do it in small volumes (but take care to have everything mixed nicely on the bottom of the tube not all the components sitting separately on the walls of the tube ) You know what you can do with your supervisor: do it like you in one tube and like her in parallel also and see what works. Only like this you can prove her when she is not right. Also it is a good practice for you as a master student to compare different protocols and choose your own later in life. With cloning is like cooking...a bit of that and a bit of the other thing, while respecting certain rules. This is why people have so many different ways of doing things.

With electroporation: I am not happy with electroporating ligation reactions. It works, but ligation reactions are highly ionic meaning they produce sparks. Take care when you electroporate to use the correct voltage settings for your cuvette gap (check this and tell me what you have). Many people do it wrong here because they use 1.8 kV for all the cuvettes. Moreover, another common mistake is too electroporate too much ligation and get the spark or not a good time constant. You need >5 time constant, check it always after electroporation. It works with 4 as well, but not very well. If it is lower than 4, it comes down to praying for a miracle. Another thing: resuspend immediately in warm SOC media. SOC media contains glucose which inhibits the cells from expressing your protein which makes their life difficult. SOC media increases the transformation efficiency several times over LB media. These are all the important tricks I can think off at the moment, we see tomorrow what else I come up with when I see your protocol.

Andreea

First: do what phage said: test your competent cells with a known plasmid and count the colonies. If you don't have pUC (and you don't want to ask your supervisor), just take the plasmid with the gene from the company: as far as I remember it has a pUC origin so it is more or less the same, because anyhow you don't need to be very precise about it. I always do this + plate 10 uL, 100 uL and the rest and count all of them and then calculate the transformation efficiency:
http://www.sciencegateway.org/tools/transform.htm
(for the more lazy of us )
Then I have question: Did you do the transformation like you said or did you modify it a bit like I said?

Everything looks ok on the transformation protocol. The only things I would change:
-do the transformation with 2 uL ligation
-use SOC media instead of LB media (see reasons above) (in lack of recipe tell me and I will check my protocol book and write it down here)
-plate 100 uL and the rest from the transformation (for the rest: spin down and discard most of the media except ~100 uL in which you resuspend your pellet)
-what kind of bacteria do you use? Dh5alpha, XL1Blue, TOP10...?
-incubation at 37C for 1 hour: shaking? I always shake them with the tube in a horizontal position with a sticky tape in the cell culture shaker at 250 rpm. I have seen people doing it also with the thermomixer inclined (put smth under the back of the thermomixer for it not to be perfectly horizontal.
-wait 16 h after transformation: if you finished yesterday at 10 in the evening, before today after lunch you cannot see anything...just saying, because I know over-anxious master students who check their transformations a few hours later

After ligation and transformation, I usually keep the rest of my ligation reaction in fridge to repeat the transformation just in case... like in case I get too many colonies and I cannot pick single ones. I am mentioning this just in case you need to repeat things: you could use the ligation from Monday if you still have it.

Andreea

ascacioc on Wed Sep 12 11:32:39 2012 said:

First: do what phage said: test your competent cells with a known plasmid and count the colonies. If you don't have pUC (and you don't want to ask your supervisor), just take the plasmid with the gene from the company: as far as I remember it has a pUC origin so it is more or less the same, because anyhow you don't need to be very precise about it. I always do this + plate 10 uL, 100 uL and the rest and count all of them and then calculate the transformation efficiency:
http://www.sciencega...s/transform.htm
(for the more lazy of us )
Then I have question: Did you do the transformation like you said or did you modify it a bit like I said?

Everything looks ok on the transformation protocol. The only things I would change:
-do the transformation with 2 uL ligation
-use SOC media instead of LB media (see reasons above) (in lack of recipe tell me and I will check my protocol book and write it down here)
-plate 100 uL and the rest from the transformation (for the rest: spin down and discard most of the media except ~100 uL in which you resuspend your pellet)
-what kind of bacteria do you use? Dh5alpha, XL1Blue, TOP10...?
-incubation at 37C for 1 hour: shaking? I always shake them with the tube in a horizontal position with a sticky tape in the cell culture shaker at 250 rpm. I have seen people doing it also with the thermomixer inclined (put smth under the back of the thermomixer for it not to be perfectly horizontal.
-wait 16 h after transformation: if you finished yesterday at 10 in the evening, before today after lunch you cannot see anything...just saying, because I know over-anxious master students who check their transformations a few hours later

After ligation and transformation, I usually keep the rest of my ligation reaction in fridge to repeat the transformation just in case... like in case I get too many colonies and I cannot pick single ones. I am mentioning this just in case you need to repeat things: you could use the ligation from Monday if you still have it.

Andreea

Don't be kapput In the end, you are a master student and without correct supervision, nobody can expect that you are born knowledgeable. Even the professors have learned this stuff from somebody.

Back to your transformation:

-when you say that the control of the competent cells worked for you, how many colonies are we talking about out of how much plated? I usually get thousands of colonies from the rest, many hundreds to a few thousands for the 100 uL and ~100 for 10 uL with the pUC plasmid. If you get some colonies at 100 uL plated, this means very little transformation efficiency that would not work with the ligation
-use SOC not LB for resuspending the cells after electroporation; see above why it is so important; I can tell you that I have seen with my eyes the difference in transformation efficiencies; LB works but SOC is sometimes even 10 times more efficient. I have a friend that never gets more than 20 colonies when she plates the entire thing after transformation. SOC media is not difficult to make. You make once half a liter and you have it for forever. (if you nicely aliquot it instead of reopening 500 times the same bottle and contaminate it in between).
-shake the cells as I said: in the eppi tube, nicely closed put a sticky tape around it and put it on the shaker for the big culture, on the metallic plate, for 1 h (no matter how weird it looks: I was the only one doing this in the lab and now most of the people are doing it as well ) or in the thermomixer also a bit tilted. You need to shake it vigorously otherwise the culture is not aerated enough and the bacteria do not recover after electroporation.
-take care to nicely spread the bacteria when you plate them; take your time and go several times with the drigalsky triangle on top of the same spot (or whatever you are using) I prefer glass beads for that, but if you are not experienced and you do it too enthusiastically, the beads end up on the cover of the Petri dish and so do your cells. How do you spread your bacteria on the plate? The whitish spot appears when you don't spread them carefully.
-check your time constant after the electroporation. There is a button somewhere in the middle that you have to press, it either shows you the kV during electroporation or the time constant. See above about what a good time constant means.

Also: I was not extremely happy with your DNA yields after PCR clean-up for the insert. Maybe you repeat the PCR and do two PCR reactions which you pool together on the same column to get a better yield. How long did you do the restriction digest, and remind me which restriction enzymes do you have? Are you using KpnI? Because I told you that I am not very sure that KpnI worked for you. Also: when you do the PCR, you add the restriction sites you are using, no? In your primers do you have extra bases (usually 6) in the extremities of the restriction sites because the enzymes need to sit on smth while cutting ?

Andreea

I can't emphasize enough how important it is do know the efficiency of your transformation. This is by far the most common problem when people have "ligation" issues. I agree about SOC. The way I do the outgrowth is to resuspend the cuvette contents in 1 ml of SOC and transfer into a 2 ml eppendorf tube. The 2 ml tubes aerate the culture better than the 1.6 ml tubes (in which the medium is trapped in the bottom). I put the tubes horizontal and loose in a beaker in the large shaker. This seems to aerate the cultures very well.

@phage: this is why I love this forum: you always find new ways others do stuff: nice beaker idea, better than my sticky tape that does not always stick, especially when you have 20 tubes

@Sameer: while waiting for me to check all the cloning strategy from the start you can retry transformation as phage says: with SOC and good shaking, this only if you have ligation reaction left or stuff with which you can repeat the ligation reaction quickly tomorrow. Today prepare SOC media and autoclave it

Andreea

ascacioc on Wed Sep 12 13:29:37 2012 said:

@phage: this is why I love this forum: you always find new ways others do stuff: nice beaker idea, better than my sticky tape that does not always stick, especially when you have 20 tubes

@Sameer: while waiting for me to check all the cloning strategy from the start you can retry transformation as phage says: with SOC and good shaking, this only if you have ligation reaction left or stuff with which you can repeat the ligation reaction quickly tomorrow. Today prepare SOC media and autoclave it

Andreea

what about doing like she said (which is so not gonna work; i tell you from now that nothing besides a contamination would grow on that plate until tomorrow) and prepare SOC media for next time I mean anyhow you need to autoclave it and aliquot it (in autoclaved tubes of course) and since basically your task is waiting right now, at least wait while doing something useful

You might also want to re-make your competent cells, or at least grow up a 10 ml overnight culture to be prepared to inoculate a 1 liter culture tomorrow.

For the SOC, you could aliquot first and then autoclave the aliquots in bulk. One less chance to contaminate things.

@phage: don't you first do SOB and then add glucose and Mg salts after autoclaving them separately? I thought glucose in media and autoclave are not a happy mixture.