Difference between revisions of "Part:BBa K1429002"

We ran a 1% gel of the digest before and after purification. We had a decent yield, maybe 40% of our initial digest product in the purified lanes. 

Next step: Transform the ligated plasmids into E. coli!

Background info:

• The plasmid is about 2000 base pairs (bp).  
• Our PCRed GFP (gene of interest) is about 750 bp.
• Assume that 1 uL of this PCR purified GFP = 50 ng (info from Ellen).

Calculations:

We have 6 uL of our gene of interest in a solution totaling 20 uL.

So we have:

\(\displaystyle \frac{6\mu L }{1}PCR \; purified \; GFP * \frac{50 ng}{1 \mu L} * \frac{1}{20\mu L} = \frac{300 ng}{20 \mu L} PCR \; purified \: GFP = 15\frac{ng}{\mu L}PCR \; purified \; GFP\)

The iGEM kit provides a linearized plasmid backbone in a solution with a concentration of 25 ng/uL.  

In our digestion step, we used 4 uL of the linearized plasmid backbone and 4 uL of the enzyme master mix. 

So, we have:

\(\displaystyle \frac{4\mu L }{1}plasmid * \frac{25 ng}{1 \mu L} * \frac{1}{(4+4)\mu L} = \frac{100 ng}{8 \mu L} plasmid= 12.5\frac{ng}{\mu L}plasmid\)

We need to have a GFP:plasmid ratio of at least 3:1 to make sure that we have enough pieces of the gene of interest to successfully connect to the plasmid backbone.

Now, we'll use the <a href="http://nebiocalculator.neb.com/#!/" target="_blank">NEBioCalculator</a>  to get the number of moles for each (see the pictures).

So, we have:

\(\displaystyle \frac{32 . 36 \frac{fmol}{\mu L} \; GFP}{9 . 632 \frac{fmol}{\mu L} \; plasmid} = 3 . 36 \; GFP : 1 \; plasmid \gt 3 \; GFP : 1 \; plasmid\)

Therefore, we can use a ratio of  1 uL of the PCR purified GFP solution to 1 uL of the digested plasmid solution.