Team:UNITN-Trento/Notebook/Labposts/06/55

From 2013.igem.org

{ "date" : "2013-06-25", "author" : "emil", "title" : "Purification and digestion of R0010", "content" : "I purified, and then quantified, 3 out of 5 of the previous inocula following the miniprep protocol with vacuum.

Quantification results
SampleQuantity
1396.1ng/µl
2523.1ng/µl
3171ng/µl
Afterwards, I digested 800ng of each sample with Spe1-HF and Pst1-HF following the digestion protocol for screening. Finally I ran the products on a gel (1,5% agarose, due to the short length of the insert: only 200 bp + prefix and suffix). I loaded 12µl of a ladder 100bp prepared as follows:
Ladder
Quantity
Water4µl
Sharpmass Euroclone 100bp ladder 1µl
glicerol solution(30%)1µl
This time, I loaded 12µl (10µl of sample and 2µl of glicerol solution 30%) of each sample.
Gel order
SampleWell
2µl loading dye1
Ladder2
sample 13
sample 24
sample 35
sample 4(Bruno e Fabio)6
sample 4(Bruno e Fabio)6
Ladder 1000 kb7
loading dye8
We can see 2 bands: the highest is the backbone (pSB1A2: 2079bp) and the lower is pLac (nearly 243bp, located between the bands 300 and 200bp of the ladder).
After the screening I digested 3µg of the pSB1A2+R0010 (sample #2) with SpeI and PstI-HF o/n following the usual protocol.", "tags" : "Plac" }