"
Team:Warsaw/Calendar-Stage1/5 July 2010
From 2010.igem.org
05.07.2010 Monday
Checking correctness of biobricks from 2010 distribution [Kasia, Jarek]
Checking: GFP-terminator=sample1, J61100=sample2, J61101=sample3, J61107=sample4, J61117=sample5, J61127=sample6 [Anderson's RBSes], B0030=sample7, B0031=sample8, B0032=sample9, B0033=sample10, B0034=sample11 [community RBSes], J23100=sample12 [synthetic promoter]
1.Finish alkaline lysis no3.
2.Run 5ul of undigested plasmids on the agarose gel.
[tu powinien byc zalacznik w postaci obrazka]
Correct amount of plasmids, expected size.
Cloning GFP-terminator behind RBSes - approach 1 -Anderson's RBSes [Ania S., Ania P., Ania O., Jarek, Kasia, Michal]
1. Rescue samples from alkaline lysis no.2 by adding RNAse.
2. SpeI/PstI digest of samples 2-11 (RBSes: J61100=2 J61101=3 J61107=4 J61117=5 J61127=6 [Anderson's RBSes], B0030=7 B0031=8 B0032=9 B0033=10 B0034=11 [community RBSes]). time:3h
3. XbaI/PstI digest of sample 1 (GFP+terminator). time:3h
4. Run GFP+terminator on agarose gel [tu powinien byc zalacznik w postaci obrazka] Small amount of plasmid.
5. Gel extraction of GFP+terminator.
6. Thermal inactivation of samples 2-6 (Anderson's RBSes) after SpeI/PstI digest.
7. Ligation of digested samples 2-6 (Anderson's RBSes) with gel-extracted GFP-terminator
8. Transformation with the ligation mixtures.
Cloning GFP-terminator behind RBSes - approach 2 [Ania S., Ania P., Ania O., Jarek, Kasia
]
1. Set up overnight GFPterminator digest with XbaI/PstI. [16ul DNA from alkaline lysis no3, 1ul XbaI, 1ulPstI, 4ul BamHI buffer, water up to 40ul.]
Prepare glycerol stocks from samples 1-12 [Ania S., Ania P., Ania O., Jarek, Kasia]
1. Set up 2 ml liquid culture of each biobrick.
