Restriction Enzyme Digestion and Phosphorylation

 

This week of the linear plasmid project, we moved into doing the restriction enzyme digestion and phosphorylation. Before I started on the digestion protocols, I nanodropped the three successful pcr tubes from last week. 

The tet sample measured at 513.4 ng/μl. The two right samples measured at 369.8 ng/μl and 428.4 ng/μl. 

To begin with the digestion protocols, we first had to get 1 μg of each samples. Going off of our previous nanodrop measurements, we got 2.5 μl of left, 2.2 μl of tet, and 2.5 μl of right. After that, we set up each tube with its specific components. 

	Left	Tet	Right
DNA	2.5 μl	2.5 μl	2.2 μl
10x rCutSmart Buffer	5 μl	5 μl	5 μl
BamHI-HF		1 μl	1 μl
Xhol	1 μl	1 μl
PCR water	41.5 μl	40.5 μl	41.8 μl

After that we incubated all of the tubes for 15 minutes at 37°C. 

We then heat inactivated the two tubes containing the Xhol enzyme (the left and tet tubes) at 65°C for 20 minutes. Bam did not need to be heat inactivated. 

After Digestion was completed we nanodroped the samples. Left measure in at 93.3 ng/μl, tet at 27.4 ng/μl, and right at 28.8 ng/μl. 

After nanodroping those samples we moved on to phosphorylation. Just like digestion we added the various components to their respective tube. 

	Left	Tet	Right
Digested DNA	7 μl	32 μl	34 μl
T4 PNK reaction buffer	5 μl	5 μl	5 μl
ATP (10 mM)	5 μl	5 μl	5 μl
T4 PNK	1 μl	1 μl	1 μl
PCR water	32 μl	16 μl	5 μl

We then incubated at 37°C for 30 minutes, then heat inactivated all three at 65°C for 20 minutes. 

Next week we will do ligation, then PCR to see our results in a gel.