Today in the Lab

Tammy

Digestion Step 1 of pSSA14 with PstI

Digestion Reaction components	Vol (μl)
H2O	4
Buffer 3	1.5
BSA	1.5
PstI	1
DNA template	7
Total	15

Digestion Step 1 of OB with PstI

Digestion Reaction components	Vol (μl)
H2O	5
Buffer 3	1.5
BSA	1.5
PstI	1
DNA template	6
Total	15

Digestion Step 2 of pSSA14 with ClaI

Digestion Reaction components	Vol (μl)
H2O	9.5
Buffer 4	3
BSA	1.5
ClaI	1
DNA template	15
Total	30

Digestion Step 2 of OB with ClaI

Digestion Reaction components	Vol (μl)
H2O	9.5
Buffer 4	3
BSA	1.5
ClaI	1
DNA template	15
Total	30

Chris

PCR Confirmation

Ran all 5 PCR product samples on a 1% gel for 40 minutes at 80 V

Desired band appeared on gel; PCR amplification was successful.

Used PCR cleanup kit to purify samples

Measured absorbance of PTP2 samples

Digestion of PTP2

Digested PTP2 with BamHI and XhoI; ran 5 tubes in parallel. Incubated at 37 C for on hour. Ran one water control.

Used PCR cleanup kit to purify digestion products. Measured absorbance to determine concentration.

Concentrations too low for ligation.

Measured absorbance of pSSA42 gel extraction products from digestion.

Gel Extraction

T123 amplification product was run on a gel and extracted.

24 gel extraction were performed in total, care was taken to minimize UV exposure (samples were not exposed for longer than 20s)

Gel of extracted PCR amplification product

Expected bands were seen, the T123 is good to go.

0A and 0B absorbances

absorbances of 0A and 0B indicated very good concentrations

Ligation and digestion

Digestion (with SphI and PstI) of T123 0A and 0B were all performed and left overnight