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minimum amounts of insert and vector for ligation - to have a successful cloning? (Mar/10/2010 )

Hi all,

for some reason my gel extraction kit (not to mention the company name) doesn't give me a high yield and I end up having very low concentrations of vector and insert.

I know that the majority of you run digested PCR product and vector on gel to determine the needed amount for ligation, but I use Nanodrop to skip electrophoresis.

my digested vector concentration today was = 26 ng/ul
my digested PCR product concentration today was = 13 ng/ul

so for a 1:1 ratio according to pmol end, and for a total volume of 20 ul I used

10 ul (260ng) vector
7.5 ul (98ng) PCR product
2 ul Buffer
1 ul T4
0.5 ul water

Is this enough?

-Curtis-

Curtis on Mar 10 2010, 06:59 AM said:

Hi all,

for some reason my gel extraction kit (not to mention the company name) doesn't give me a high yield and I end up having very low concentrations of vector and insert.

I know that the majority of you run digested PCR product and vector on gel to determine the needed amount for ligation, but I use Nanodrop to skip electrophoresis.

my digested vector concentration today was = 26 ng/ul
my digested PCR product concentration today was = 13 ng/ul

so for a 1:1 ratio according to pmol end, and for a total volume of 20 ul I used

10 ul (260ng) vector
7.5 ul (98ng) PCR product
2 ul Buffer
1 ul T4
0.5 ul water

Is this enough?



NEB T4 ligase recommends using 1-10 ug/ml of DNA for circular formation. You've got about 360 ng in 21 ul, so that's about 17-18 ug/ml. You've got plenty.

question 7: http://www.neb.com/nebecomm/products/faqproductm0202.asp#346

-fishdoc-

I get worse yields than that sometimes for gel extraction! I've successfully ligated negative yields of vector and insert together and gotten plenty of colonies though.

-microgirl-

thanks a lot....

I don't know what's wrong with my gel extraction kit, but last time I used Qiagen and it was fine. I got over 100-200 ng/ul.

Today I loaded 4 ug of plasmid on the gel, but after extraction I only managed to purify 700 ng in total. The rest is gone.

-Curtis-

Curtis on Mar 10 2010, 08:21 AM said:

thanks a lot....

I don't know what's wrong with my gel extraction kit, but last time I used Qiagen and it was fine. I got over 100-200 ng/ul.

Today I loaded 4 ug of plasmid on the gel, but after extraction I only managed to purify 700 ng in total. The rest is gone.


Wow that's a lot. I usually use 0.5-0.75 ug for a digest which I exert from the gel. But 4 ug seems huge. I never measured the amount of DNA after Gel Ex and basically just determine the ratio by visually checking the bands of a 2-3ul or eluate on the gel. Works perfect for me.

-snoopyx-

Hello,

i also nanodrop like you and i found that a 1:3 ratio of vector to insert works well. I usually do 50ng of vector and my vector concentrations are usually 20ng/ul only.

just curious, when you nanodrop, does the histogram of absorption vs wavelength give you only one peak or do you have multiple peaks or extra "bumps" ?

-molstudent-

it is just one big peak, but it doesn't look very smooth some times.

-Curtis-

I would try a ligation with 10-20% of the DNA in your current reaction. The reason is not that there is too much DNA, but that there may be too much other junk in your DNA extraction, which can be diluted out in the ligation reaction with water. Doing reactions like this, where virtually all of the volume comes from the input DNA is risky when the purity of the extracted DNA is unknown.

-phage434-