Team:UCLA/Notebook/Spider Silk Genetics/8 May 2015

iGEM UCLA




5/8/2015

5/8/2015


1 T4/T7 Ligation Test

We tested the ability for the BsaI digested and gel purified monomers to ligate together. We ran these initial tests using both T4 and T7 DNA ligase using the following protocols (50 ng for each DNA species):

1.1 T4 Ligation

10x T4 Ligase Buffer1.5 uL
2AB 4 uL
2CA 5 uL
T4 Ligase 1 uL
ddH2O 3.5 uL
Total 15 uL
25 C 10 min

1.2 T7 Ligation

2x T7 Ligase Buffer7.5 uL
2AB 4 uL
2BC 5 uL
T7 Ligase 0.5 uL
ddH2O 0 uL
Total 17 uL
25 C 10 min

2 Results

We cast a 2% TAE gel to visualize the results. We use 2 uL of NEB 50 bp ladder. The gel was run at 150V for 35 min.

PIC

Figure 1: Preliminary ligation test with T4 and T7 polymerase. The band slightly larger than 200 bp is our desired ligation product.

Our ligation test indicates that the new sticky ends exhibit specific binding. In both reactions, the ligation was incomplete due to the presence of a band at 100 bp. This may be due to non-equal amounts of DNA.

We plan to conduct this same test next week using all possible combinations between the three monomers to verify specific binding. We plan to test both T4 and T7.