• EF colonies present! So, we took 1ml off for further growth and miniprepped the remaining 4ml in triple
• Nanodrop concentrations:

• We performed a digest with EcoRI and SpeI to cut out the insert and check it's length. Enzymes were added separately, with 1h in between. Total volume was 30µl.
• Calculations restriction

• In the afternoon, the remaining 1ml was re-plated and put on 37°C

2)

• Test 1: Results from yesterday's restriction test were inconclusive, because the DNA ladder didn't run properly so the length of the fragments couldn't be read. Therefore, we did it again...
• Restriction=OK! Test 1 completed.

3)

• Test 2: cutting with 2 enzymes subsequently (1h incubation in between) in a volume of 30µl. Using a larger volume should dilute the glycerol, which inhibits cutting. After that, the products are incubated overnight to allow full restriction. sam8 and fcs are cut with EcoRI and SpeI; sam5 and ech are cut with EcoRI and XbaI

• Calculations restriction

• Restriction digest of X, Y and with EcoR1 and Pst1
• Gelelectrophoresis to check fragments and gelextraction
• Fluid medium from outplated cells (overgrowth)
• TOPO-cloning (with new protocol from Invitrogen) for W and Z