{ "date" : "2013-08-02", "author" : "thomas", "title" : "pSpac + GFP screening", "content" : "Today I started from the overnight cultures that I did yesterday. I performed a miniprep purification following this protocol. I quantified then the products at the nanodrop obtaining these results:

Quantification results
pXyl-GFP 1132.7 ng/µl
pXyl-GFP 2102 ng/µl
pXyl-GFP 3126 ng/µl
pSpac-GFP 1:1 1198 ng/µl
pSpac-GFP 1:1 2322 ng/µl
pSpac-GFP 1:4 1154 ng/µl
pSpac-GFP 1:4 2249 ng/µl
pSpac-GFP 1:4 3196 ng/µl
I proceeded then with a screening digestion in order to confirm the products. Kapa universal Ladder was adopted. Lane 2,3,4: as you can see, the bands confirmed the reamplification of BBa_K823024 (4974 bp) + BBa_E0840 (878 bp).Lane 5,6,7,8,9: as you can see, the higher band confirmed the vector BBa_K823026 (8289 bp) while the lower band didn't confirm the insert BBa_E0840 (878 bp). I think it took a fusion between BBa_E0840 (878 bp) and RFP coding device (1069 bp) since the band is at the level of 2100 bp and since I had few pellets that were red. So the ligation between pSpac and GFP failed.. I hope that the next time I'll be more lucky! ", "tags" : "pSpac-GFP-pXyl" }