20 replies to this topic

[netnus]

Hi,
  I am trying to ligate my gene 400bp to a PET9a (4kbp)vector. I double digest the pcr and vector with Nde1 and BamH1. In order to avoid the star activity, I first digest with Nde1 and inactivate Nde1 at 65 degree for 10 min. Then I digest with BamH1 overnight (about 16h). After that, I purify the cut pcr and vector for the agarose gel with Qiagen kits.
  I then do the ligation with pcr:vector=3:1. The T4 ligase I use is from Fisher Scientific. The protocol for the ligation is room temperature 3h. To make sure the ligation occurs,I take 15-20ul ligation mix to run on the gel.
The gel shows 3 bands: one around 4k I regard it as unreacted vector,one around 5k,one around 1k.
  I then take 5ul ligation mix to do  the transformation. the competent cell is XL-10 gold from strategene. I spread 100ul and 150ul on the LB-Kan plate, but not colony appears at all.
  Can anyone give me some suggestions? Do you think the ligation works? or any problem in the transformation?
  Thanks!!

  netnus

[ravibiot]

Hi

May be you can check for linear ligation of PCR digest (since, your PCR product size is 400 bp and vector is 4 kb. So, the expected ligation product should be at 4.4 kb)
I suspect the problem lies in ligation. I used to do the ligation reaction at 4 C for 14-16 h (undisturbed), and i  get efficient ligation. For transformation i take only 2 ul ligation product. I hope you will get good transformants.

Best regards,
ravi
INDIA

[popogirlxd]

[QUOTE]:I used to do the ligation reaction at 4 C for 14-16 h (undisturbed)

I know 4c is the best for ligase activity, but  isn't  the T  too low for the vector and insert thermal movement? May I ask you what ligase you use?

Thanks!

[Janina]

Hace you tested your cells (efficiency, positive control)? Have you plated a negative control (religation)? Have you used new LB-Kan-plates? Someone in this forum wrote that Kan is reduced faster than "normal" antibiotics...

I use only 2 ul for transformation, too. The more is often not the best... especially in mobi...  

[netnus]

Janina, on Dec 1 2004, 01:59 AM, said:

Hace you tested your cells (efficiency, positive control)? Have you plated a negative control (religation)? Have you used new LB-Kan-plates? Someone in this forum wrote that Kan is reduced faster than "normal" antibiotics...

I use only 2 ul for transformation, too. The more is often not the best... especially in mobi... 

Yes! I do the positive control and negative control. The positive control is ok while the negative control shows nothing. So the double digestion may be ok.
  
And if you use 2 ul for transformation. Do you spread all the cells to the LB plate by concentrating down to 100 ul?

Thank you for your help!

[Janina]

I have 100 ul competent cells, fill them during the protocol with 400 ul LB and plate 50 and 150 ul.

[netnus]

ravibiot, on Nov 30 2004, 09:22 PM, said:

Hi

May be you can check for linear ligation of PCR digest (since, your PCR product size is 400 bp and vector is 4 kb. So, the expected ligation product should be at 4.4 kb)
I suspect the problem lies in ligation. I used to do the ligation reaction at 4 C for 14-16 h (undisturbed), and i  get efficient ligation. For transformation i take only 2 ul ligation product. I hope you will get good transformants.

Best regards,
ravi
INDIA

Yes! I just follow your instructions. I incubate the ligation at 4 degree overnight (~16h). I try the transformation again,but still nothing grows.
I even change the ligase from Fisher scientific to Promega ligafast. In promega's protocol,the ligation takes only 5 minutes under room temperature. I don't think there is any problem in the transformation since the positive control grows ok!

Need help,please!

Thanks

netnus

[Janina]

Although protocol tells 5 min at RT, I do ligate with such fast kits up to 15 min... It is also necessary to mix/vortex reaction (and spin down) before incubating.

Perhaps in your calculation something  is wrong... Did you measure conc by photometer?

[netnus]

Janina, on Dec 2 2004, 09:18 AM, said:

Although protocol tells 5 min at RT, I do ligate with such fast kits up to 15 min... It is also necessary to mix/vortex reaction (and spin down) before incubating.

Perhaps in your calculation something  is wrong... Did you measure conc by photometer?

Yes! I do votex and spin down before they are incubated. I use UV-photometer to estimate the conc.
Seems like the problem is the ligation. Usually how long  would you like to double digest your circular vector and your linear insert? Someone just suggests me to shorten the digestion time to 2 h for cVector and overnight for linear insert.

  Really want you can help me out! Greatly appreciate!


  netnus

Edited by netnus, 02 December 2004 - 12:28 PM.

[sean888]

I use ligation condition: temp 16 for 16hr, and take 5 for transformation, the result is good, I am detect the bio-activate of the gene now.

[Janina]

I do a digest at 37 °C for 3 h, 10 min aT 65 °C... Perhaps it is better to do a sequential digestion.
But I make no difference in incubation time between vector and insert.

Edited by Janina, 03 December 2004 - 01:51 AM.

[caro]

Hi

It has been some while since i made ligation and transformation, but have you calculated the ratio vector:insert in grams or in molar??? There is a big difference when they have such a great difference in size.
I think it should be in molar (=number og molecules)

Regards Caro

[netnus]

caro, on Dec 3 2004, 04:12 AM, said:

Hi

It has been some while since i made ligation and transformation, but have you calculated the ratio vector:insert in grams or in molar??? There is a big difference when they have such a great difference in size.
I think it should be in molar (=number og molecules)

Regards Caro

I try ligation with the ratio based on the molar ratio. I have ever tried the ration in grams. But neither works. If the ratio is in molar,only a very small amount of insert has to be added. If the vector is not completely double cut, is there much more chance for vector to self-circularize?

Edited by netnus, 03 December 2004 - 06:40 AM.

[frà]

Hi guys,
I'm not sure about the amunt of DNA to do tarsformation because when I do trasformation to do for example maxiprep ( in any case not DNA from ligation ) I used 1 microgram of DNA and 50 ng of DNA to compare the efficiency.when I use 1 microgram the efficiency is more high than 50 ng.
I think the limit of amount of DNA is for ligation. Don't you think?
I'm sorry for my english.

[praveen_iitb]

HI ,
To standardise the ligation reaction one easiest step u can follow is
digest ur plasmid with one of ur enzyme and ligate back  in diffrent conditions
and look for colonies .
perform a transformation with these contols
1) uncut plasmid
2) single cut plamid
3) single cut plamid + ligase
this approch at least will allow to check where is the problem??

bye
praveen