Biotech Lab Report 4

Posts : 107 Join date : 2009-10-22

Should the transformation efficiency of the uncut DNA be larger or smaller than the recombinant DNA? I am confused because we got no white colonies and tons of blue colonies on our ligated DNA plate. We had less colonies on our uncut plate. I am thinking it should have been the other way around if our experiment worked right.

Posts : 100 Join date : 2009-10-22

truly cant answer that yet.
we got 48 blue & 6 white colonies on plate 2, total of 54 CFU
and 51 blue & 0 white colonies on plate 4, total of 51 CFU

Posts : 100 Join date : 2009-10-22

I think the answer to your question really has to do with the # of bacterial cells plated. Calculation from the concentration used on the plates. Uncut - 0.004 ug would not yield the same amount as 0.042 ug in the Ligation. Those aren't the correct figures. That's concentration & we used 3 ul of each.

Posts : 107 Join date : 2009-10-22

For the Trans Eff.

Take 0.042 ug/ul DNA X 3 ul = 0.126 ug

You plated 200 ul/1000 ul = 0.2

Multiply .2 X 0.126 = 0.0252 ug

take your cfu/0.0252 ug to get Trans. Eff for the ligation.

The other is the same except the DNA is
0.024 ug/ul X 3 ul = 0.072 ug

Multiply this by .2.

Divided cfu by this number.

Posts : 107 Join date : 2009-10-22

Looking at your numbers, ours are way different. We had 314 total colonies on the ligation plate (all blue) and only 65 colonies on the other plate (all blue).

Posts : 100 Join date : 2009-10-22

a really good site that lists things that affect Transformation Efficiency:
http://www.sigmaaldrich.com/life-science/molecular-biology/cloning-and-expression/learning-center/transformation-efficiency.html

Posts : 69 Join date : 2009-10-22

Our ligated DNA transformation eff. was less than the no insert and pBLU.

Are the pBLU and TE considered negative or positive controls? The definitions for them online are confusing as hell.

Posts : 107 Join date : 2009-10-22

Did you have growth on your no insert LB/Amp plate Kyle? We had none, so had no transformation efficiency for that plate.

The TE is a negative control.
The uncut pBlu is a positive control.

Posts : 69 Join date : 2009-10-22

That's what I figured. And the LB plate is a negative control too?

Our no insert lb/amp plate had blue colonies on it, and we did calculate its trans eff.

Posts : 69 Join date : 2009-10-22

Wait I think I did my ligation efficiency wrong. I was supposed to add the pBLU and pKAN values right? The 0.0144 and 0.0108? And divide that into my CFU?

Posts : 69 Join date : 2009-10-22

Wait, why the hell do I have white colonies (one) on my pBLU plate? Isn't that a control without any inserts? DID MY BACTERIA MUTATE OMG

Posts : 107 Join date : 2009-10-22

See my above calculations that I showed Lisa. That is how I did trans. eff.

Posts : 107 Join date : 2009-10-22

I just got a reply from Dr. Bradshaw and she said that the uncut pBlu should have had the highest transformation efficiency, so mine data is just horrible.

Posts : 69 Join date : 2009-10-22

Yeah but for the ligation you need to calculate the concentration for the dna in both pblu and pkan, and add them together for the bottom part of the efficiency fraction, right? And then the No Insert is just the pblu portion?

Posts : 100 Join date : 2009-10-22

this is what I got

Ligation
Number of Colony Forming Units on the whole plate were counted = 54 CFU
1.      Known quantity of DNA added = 0.042 ug/µl* x 3 µl = 0.126 µg
2.      Fraction of DNA plated = 200 µl/ 1000 µl = 0.2 = 20%
3.      Total DNA plated = 0.126 µg x 0.2 = 0.0252 µg
4.      Transformation Efficiency = #CFU / µg of DNA added = 54 CFU / 0.0252 µg =
2.14 x 103 CFU/µg DNA

No Insert
Number of Colony Forming Units on the whole plate were counted = 51 CFU
1.      Known quantity of DNA added = 0.024 ug/µl** x 3 µl = 0.072 µg
2.      Fraction of DNA plated = 200 µl/ 1000 µl = 0.2 = 20%
3.      Total DNA plated = 0.072 µg x 0.2 = 0.0144 µg
4.      Transformation Efficiency = #CFU / µg of DNA added = 51 CFU / 0.0144 µg =
3.54 x 103 CFU/µg DNA