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Ligation of large ammount of insert (no transformation)

ligation

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7 replies to this topic

#1 Eddie*

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Posted 18 February 2013 - 01:55 AM

Hey guys,

I need to get large amounts of my vector but cannot transform it in E. Coli (due to some special bases), so i needed to optimize my PCR, Digestion and ligation. Now I am currently trying to optimize my ligation.

My Insert is about 500bp big, my vector 5590bp.

I use a mol-ratio of insert:vector 3:1

So i now want to ligate:

1µg Insert
4µg Plasmid

To get the total DNA conentration of about 10µg/mL I want to use a total volume of 500µL and 3µL of T4 DNA Ligase (NEB)

Now my questions ;)

Do you have any experiences ligating large amounts? Do you think 500µL is a volume big enough? How much T4 Ligase would you use?

thans a lot in advance :)

#2 neuron

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Posted 18 February 2013 - 03:27 AM

500 ul is really huge, I mean for ligations generally it is said that minimum volumes should be taken. Otherwise your ratios of insert and vector look fine. How much T4 DNA ligase you will use? 50ulPosted Image ...No experience of setting up this big ligationPosted Image but at least 15ul should be usedPosted Image ,....good luck Posted Image

#3 phage434

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Posted 18 February 2013 - 06:05 AM

I think you will be disappointed. Ligation is not very efficient, which is why we use highly competent cells to select the few correct ligations. You will have a hard time getting good yield, and it will be difficult to tell how good a yield you actually achieve. You probably don't need much ligase -- its standard concentrations are 100x - 1000x more than necessary.

#4 Eddie*

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Posted 18 February 2013 - 10:11 AM

Thanks for your responses.

I set it up and we'll tomorrow what happend :)

#5 bob1

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Posted 18 February 2013 - 11:57 AM

I use a mol-ratio of insert:vector 3:1

So i now want to ligate:

1µg Insert
4µg Plasmid

Note that this isn't a molar ratio - it is a ug amount!

#6 neuron

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Posted 19 February 2013 - 03:12 AM

You are right bob but I think after molar calculations and based on the size ratio of vector and insert Eddie will have to take this ratio of amountPosted Image

#7 GNANA

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Posted 19 February 2013 - 03:31 AM

I always run gel on one microlitre of vector and insert along before setting up ligation and based on the band intensity (purely manual) i calculate the 1:3 ratio. for instance if my vector size is 4500 and insert is 1500 bp the equal intensity of both the bands means they already are in 1:3 molar ratio. so i take 1 microlitres each and set the ligation. this never let me down untill now.

Edited by GNANA, 19 February 2013 - 03:33 AM.

I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#8 Eddie*

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Posted 21 February 2013 - 05:08 AM

So just to let you know...it didn't work ;)

I'll continue screening :)





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