2) Restriction digest of the DNA from step 1 with EcoRI and PstI

3) Gel electrophoresis to check if the insert in the plasmid is the correct size, which it was, so the ligation was succesful.

• Cells didn't grow. Yet again...
• Re-plated the ligation products (SAMS and EF)

• We propose 3 tests to check if the restriction digest (esp. between EcoRI and XbaI, because they lie very close together) works properly:

Test 1: Cut every plasmid (sam8, sam5, ech, Fcs) separately with EcoRI, XbaI and -only sam8 and fcs- with SpeI.

Test 2: Cut subsequently with EcoRI and XbaI, leaving an incubation period of 1h between two digests. Total volume: 30µl, to dilute glycerol (which inhibits restriction)

Test 3: Use pUC18 vector

• Made fluid cultures of sam8, sam5, ech, fcs

• PCR purification miniprep and nanodrop of R on mutation 2
• Start ethanol precipitation (overnight ethanol + sodium acetate) to create a better result (more purified product) of product R with mutation 202
• PCR of the fragments of TOPO (W and Z) was done to check if TOPO worked. Gelelectrophoresis showed nothing so TOPO cloning failed again for virA.