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Ligation Efforts Continue

  • In addition to trying the triple-ligation strategy started on 7/7 (ligation attemp #3) will try trouble-shooting the ligation strategy used in attempts #1 and #2. In particular, will modify digestion protocol and test EcoRI and XbaI (older enzymes) for activity.

Ligation Attempt #3 (Triple Ligation)

Ran triple ligation all day at room temperature and stored at 4°C. Ligation contents were as follow to have a 1:1:1 stoichiometric ratio of all three components.

Component Volume
Distilled Water 6.4 μL
T4 Ligase buffer 10x 2 μL
NEB T4 Ligase 1 μL
pSB1C3 solution 2 μL
B0015 solution 2.4 μL
phsABC solution 6.2 μL
Total 20 μL

Ligation Attempt #4

Concerns about enzyme activity led to reversing the order of the serial digestion of B0015 background so that the first digestion is XbaI (low salt buffer) and the second is EcoRI (high salt buffer). Will also omit the pre-ligation PCR purification step.

Overnight digestion of ligation components

Set up the following digestions to run overnight at 37°C. The EcoRI was newly purchased as there was concern about the age of the previously used enzyme.

phsABC double digestion
NEB EcoRI Buffer 5 μL
BSA 100x 0.5 μL
phsABC solution 30.8 μL (1 μg DNA)
NEB EcoRI 1.8 μL
NEB SpeI 1.8 μL
Sterile H2O 10.1 μL
B0015 digestion
NEB Buffer 4 5 μL
BSA 100x 0.5 μL
NEB XbaI 3.6 μL
B0015 solution 3.8 μL ( 1 μg DNA)
Sterile H2O 37.1 μL

Wetlab work for this day is also recorded on pages 52 and 53 of the hard copy lab notebook.