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#1 siddharthsameer

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Posted 07 September 2012 - 02:38 AM

Hello everyone
I need suggestion and advice for the ligation
I want to ligate 1,4 kb insert with the vector pPICZaplha A(3,6kb) and transform into E.coli competent TOP10: agter the gel extraction the concentration of my PCR product (insert) is 9,15ng/µl and for the vector is 30,5ng/µl. Are my concentration fine to carry out the ligation?
I am here to take the advice because last time I had more or less the same concentration my both insert and vector but after the transformation I didnt get any colobnies..So can anyone suggest me the correct manner to carry out the proper ligation.. i am stuck up with the 4 months.. Kindly suggest me how should I carry it out to get the result.
Last time i had the molar ration of 2:1,3:1,4:1 but noone of them worked..
Waiting for the quick reply thanks and regards

#2 ascacioc

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Posted 07 September 2012 - 03:55 AM

It is a bit too low for my taste but it should work:
take 8 uL of your insert; 0.5 uL of your backbone; 1 uL of 10X T4 DNA ligase buffer, 0.5 T4 DNA ligase (if NEB otherwise tell me the company you have and I check their recommendations and come back to you). I incubate this at room temperature for 1 hour. Then transform 4 uL of this in my competent cells if chemical competent cells and 2 uL if electrocompetent and plate 100 uL of the transformant and 900 uL of the transformant (spin down, throw away most of the liquid except for ~100 uL that you use for resuspending the cell pellet) on 2 agar plates and grow them ON at 37oC (minimum of 16 h). Colony PCR tomorrow.

Would you mind sharing with me your transformation method for me to check some things?

Andreea

#3 phage434

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Posted 07 September 2012 - 04:51 AM

Could you tell us how you have prepared your plasmid and insert? Usually "ligation" problems are really problems with the insert and vector preparation, or with transformation.

#4 siddharthsameer

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Posted 09 September 2012 - 02:24 AM

It is a bit too low for my taste but it should work:
take 8 uL of your insert; 0.5 uL of your backbone; 1 uL of 10X T4 DNA ligase buffer, 0.5 T4 DNA ligase (if NEB otherwise tell me the company you have and I check their recommendations and come back to you). I incubate this at room temperature for 1 hour. Then transform 4 uL of this in my competent cells if chemical competent cells and 2 uL if electrocompetent and plate 100 uL of the transformant and 900 uL of the transformant (spin down, throw away most of the liquid except for ~100 uL that you use for resuspending the cell pellet) on 2 agar plates and grow them ON at 37oC (minimum of 16 h). Colony PCR tomorrow.

Would you mind sharing with me your transformation method for me to check some things?

Andreea

Hello andreaa
sorry for the late reply as my internet is kaputt Posted Image.. I am using T4 DNA ligase from NEB, I dont mind sharing my transformation protocol but i can only send it to you once I go to the lab tomorrow, i will attach and send it to you... You know my supervisor is asking me to make 40microlitre total volume for the ligation as the last time.and asking for the incubation overnight,, i am very skeptical about that...But i do the transformation through electroporation...
sorry for being late in r

Edited by siddharthsameer, 09 September 2012 - 02:26 AM.


#5 siddharthsameer

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Posted 09 September 2012 - 02:25 AM

Could you tell us how you have prepared your plasmid and insert? Usually "ligation" problems are really problems with the insert and vector preparation, or with transformation.

Hello
I prepared my vector through midi prep and the inser was order from the company.... my vector is 3.6kb and insert is 1.4kb..

#6 ascacioc

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Posted 09 September 2012 - 02:36 AM

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

#7 phage434

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Posted 09 September 2012 - 06:32 AM

You said where your DNA comes from, but not how it is being cut and purified. These are the critical steps, and are often where the problem lies.

#8 ascacioc

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Posted 09 September 2012 - 06:36 AM

@phage: We have already cut the DNA 2 weeks ago in another thread :).

http://www.protocol-...hl__+midi +prep

Except that I think his KpnI is dead or smth is wrong with it, the rest looked fine :) But it does not hurt to have a second and third opinion. Especially when it is your opinion. :)

Andreea

#9 siddharthsameer

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Posted 09 September 2012 - 11:49 PM

Do not worry Posted Image You are so panicky Posted Image 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 Posted Image ) 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

hello andreaa
this is my transformation protocol into competent cells
1, ligation mixture, competent cell on ice provided with 10ml lb media
40µl of competent cell and 1-2µl ligation mixture mixed together and put on ice for 1 minute
micropiulser on the program with 0,2cm cuvette, 40µl cell suspension 2,5 kv, 5 ms
transfer solution into the uvett
add 1ml lb media andtransfer the solution into eppi on place on ice
subsequent incubation at 37 for 60 min
plating the solution 20µl 100µl and 200µl..
this is what i follwed last time...
i also made the competent cell last month that i will be using for the transformation,, and it is electrocompetent cell as far as I think..

#10 phage434

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Posted 10 September 2012 - 12:18 PM

I would definitely test the competence of your cells. Dilute a known plasmid (pUC19 is typical) to the 100 pg/ul level, and transform with your cells and protocol, plating out on amp plates. You should get 10^9 cfu/ug of DNA (if you use 1 ul, then you should get 10^5 CFU transformed). If you plate 20 ul of a 1 ml outgrowth, then you should see 2000 colonies.

#11 siddharthsameer

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Posted 11 September 2012 - 10:13 PM

Do not worry Posted Image You are so panicky Posted Image 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 Posted Image ) 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

hello andreaa#
My ligation failed again i did not get any colony....what to do next kindly help me.

#12 ascacioc

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Posted 12 September 2012 - 03:32 AM

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

#13 siddharthsameer

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Posted 12 September 2012 - 03:53 AM

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 Posted Image)
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 Posted Image

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

I followed exactly what you said for the transformation, i spined it down and transformed 100µl... however the control also worked for me. I kept for incubation at 37 without shaking.. i am using TOP10 for the transformation...I plated yesterday at 5 and till now on colony is seen..but i could see a small spot of in one of the plate in which i suspended with 100µl.. but i don thnk that is my colony... everything from pcr till ligation was in ordunung i really dont know what exactly happend...I used biorad micropulser and just after the click sound i added 1ml lb media nad incubated for 1hrs or so....ich bin kaputt :(

#14 ascacioc

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Posted 12 September 2012 - 04:45 AM

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

#15 phage434

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Posted 12 September 2012 - 05:01 AM

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.




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