"
Team:HKU HongKong/Data/Weekly Notebook.html
From 2012.igem.org
Team:HKU HK
From 2011.igem.org
Daily Lab Notebook
Weeks 1&2:
1)
Amplification of pvdQ gene using PCR from Pseudomonas aeuriginosa genomic DNA. The designed primers contain the prefix and suffix so that typical digestion for insertion into iGEM’s standard vector can be subsequently performed.
2)Test digestion of amplified pvdQ with EcoRI only, EcoRI & SpeI, PstI, and EcoRI & XbaI.
- Result: Two bands present in the EcoRI only digestion instead of the expected single, linear fragment. Indicates presence of a skeptical EcoRI site – a sequence that is not identical to the conventional EcoRI palindromic sequence but similar such that EcoRI restriction enzyme non-specifically cuts it).
3)
Repeat the digestion of pvdQ with EcoRI and SpeI multiple times to obtain the desirable conditions of the digestion to prevent Star Activity.
4) Mass single digestion of the amplified pvdQ with EcoRI. Desirable fragment size should be about 5,600bp. The fragments that result due to Star activity are 3,800 and 1,800bp.
4) 5). Gel purification and Nano-drop concentration check of digested pvdQ.
