Team:Stockholm/11 August 2010

From 2010.igem.org

Hassan

Started making animation for what's happening in our project.
TODO for tomorrow: complete the interaction map, and starting summarizing texts for the map, hope to finish it by sunday...

Nina

Digestion of Tyrosinase

I cut the miniprepped site directed mutagenesis and PCR product tyrosinase with the restriction enzyme NgoMIV.

Digestion:

Incubated in 37 °C for 2 hours.

I ran the digested products in an agarose 1 % gel 80 V.

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

Arrangement on gel:

I ran a new digestion since the first one did not indicate wether or not the digest had worked or not. The second digest I altered the amounts in the mixture into those values that I have used previously when digesting.

Second digest:

Incubated in 37 °C for 1 hour (PCR prod) and 3 hours (vector).

I ran the digested products in an agarose 1 % gel 80 V.

Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas

Arrangement on gel:

I still don't get reliable results that the enzyme has cut, therefore I will run a gel tomorrow with an uncut vector to compare with the cut vector sample, this should show if the enzyme can cut and from there I know that the site directed mutagenesis tyrosinase samples have been succesful in altering the restiction site for NgoMIV since they won't be cut.

Colony PCR of protein A (ZZ domain)

I ran a colony PCR on the transformed protein A ZZ domain in the bank vector C in order to verify that I have inserted the gene correct in the vector.

PCR master mix (calculated for 7 tubes):

MgCl2 7 ul
Buffer phusion 5X 70 ul
dNTP 10 uM 7ul
primer VR 10 uM 21 ul
primer Forward 10 uM 21 ul
polymerase PjuX7 7 ul
H2O 210 ul

5 ul of the PCR products with 1 ul loading dye 6X were run on an agarose 1 % gel, 80 V.

Ladder to the right: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas

Ladder to the left: GeneRuler™ Low Range DNA Ladder, 25-700 bp Fermentas

Arrangement on gel:

The lane number 5 looks good and verifys that I have protein A ZZ domain in the bank vector with chrloramphenicol, therefore I will continue with this colony (#5) when working with protein A ZZ in the bank vector.

Overnight culture of protein A (ZZ domain)

I inoculated three falcon tubes containing 12 ml LB and 24 ul chloramphenicol (50mg/ml) with the colony number 5 from the protein A ZZ domain dish. This was incubated overnight in 37 °C with shake. These samples will be miniprepped tomorrow.