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.
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 |
---|
Water | 4µl |
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 orderSample | Well |
---|
2µl loading dye | 1 |
Ladder | 2 |
sample 1 | 3 |
sample 2 | 4 |
sample 3 | 5 |
sample 4(Bruno e Fabio) | 6 |
sample 4(Bruno e Fabio) | 6 |
Ladder 1000 kb | 7 |
loading dye | 8 |
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"
}