Team:UCLA/Notebook/Spider Silk Genetics/13 May 2015

iGEM UCLA




5/13/2015

5/13/2015


1 NotI Concatemers

Used yesterday’s gel purified fragments. We performed ligation using a 5:1 ratio of insert to vector. Our vector size is 2046 bp. Our insert is 143 bp. For 1121 ng of vector, we need 391.75 ng of insert. We set up the ligation reaction as below:

Amount (uL)
Vector (112.12 ng/uL)10
Insert (52.63 ng/uL) 10
10X T4 Ligase Buffer 2.5
T4 Ligase 3
Total 25.5

We used excess insert because the recorded concentration from the nanodrop was higher than the theoretical yield. In addition, we used more T4 Ligase than in typical reactions due to the reaction having three times as many sticky ends to ligate.

2 Results

We cast a 0.8% TAE agarose gel to separate the bands. We ran this particular gel at 90 V for 2 hours. We excised four bands indicated for gel purification (Qiagen). The bottom most band may be due to vector self-ligation. The second band at 2.2 kb is probably singly ligated product.

PIC

Figure 1: Ligation of 2CA NotI digested vector and insert. We excised the 4 indicated bands for purification.

In the future, we should CIP treat the vector so it doesn’t re-ligate.