"

From 2010.igem.org

Contents 1 Andreas 1.1 CPP troubleshooting 2 Nina 2.1 Protein expression of IgG protease from pEX 2.2 PCR on Tyrosinase

Andreas

CPP troubleshooting

Analyzed sequencing results of our constructed CPPs (LMWP and Transportan 10). It seems like our target vector recircularizes instead of ligating with our CPP primers. Me, Johan and Nina are working on troubleshooting this, however it looks like we will soon have to synthesize our genes, since our primer ligations just don't seem to work.

---

Nina

Protein expression of IgG protease from pEX

BL21 cultures of IgG in pEX and bFGF in original vector from plates used to inoculate for IPTG induction:

A streak of colonies from the IgG plate was inoculated in two separate falcon tubes containing 12 ml LB and 24 ul ampicillin (50 mg/ml). ~10 colonies from the bFGF plate was inoculated in two separate falcon tubes containing 12 ml LB and 24 ul ampicillin (50 mg/ml). This was incubated with shake in 37 °C until OD reaches around 0.6.

---

PCR on Tyrosinase

I have had trouble obtaining a band representing tyrosinase on a 1 % agarose gel after running a PCR. Therefore I ran two new PCR:s, one with an annealing time of 2.5 min and another with 3 min.

1 = 2.5 min

2 = 3 min

DNA Ladder: FastRuler™ Middle Range, ready-to-use, 100-5000 bp Fermentas

It looks like I got a correct band on both samples, however the 2.5 min looks better than the other.