"

From 2013.igem.org

{ "date" : "2013-08-12", "author" : "thomas-viola", "title" : "We don't give up!", "content" : "Ok yesterday we discovered that the ligation between BBa_S04617 and BBa_K1065002 failed so today we focused on a new cloning strategy. We decided to amplify both parts by PCR following this protocol. BBa_Fwd and BBa_Rev primers were adopted. PCR products were then screened, purified and quantified at the nanodrop..

As you can see from the picture, the gel confirmed our PCR reaction since in lane 1 there is a band at heigh of 1215 bp (BBa_K1065002) and in lane 3 one band at heigh of 1245 bp (BBa_S04617). Kapa universal ladder was adopted.We proceeded then performing an o/n digestion on the following parts in order to do a tri Assembly:

Part	Enzymes used	quantification after purification
pSB1C3	EcorI+PstI	90.1 ng/µl
EFE+B0015	XbaI+PstI	104.7 ng/µl
s04617	EcorI+SpeI	133.5 ng/µl

Tomorrow we will proceed with a ligation of the three digestion products..!", "tags" : "pFixK-cI-pLambda-EFE" }