Team:UCLA/Notebook/Spider Silk Genetics/4 May 2015

iGEM UCLA





5/4/2015

5/4/2015


1 BsaI Digestion Test for 2AB

Tested digestion of 5 ug of plasmid to see efficiency of digestion.

Amount (uL)
BsaI 2 uL
2AB (287 ng/uL)14.7 uL
Cutsmart 5 uL
ddH2O 28.28
Total 50 uL
50 C 2 hr
65 C 20 min

Results are shown below:

PIC

Figure 1: Results of BsaI digestion test. The band at about 100 bp corresponds to the target digested insert.

∙ The digestion process works, so we’ll be proceeding with this protocol.

∙ We gel extracted the digested insert band. The end result was 11.9 ng/uL, A (260/280): 4.4

2 BsaI Digestion for Post-elution primer amplified MaSp

We BsaI digested the PCR products from previous PCRs using the following protocol

2.1 BsaI Digest Protocol

2AB(61.43 ng/uL)2BC* CA (56.65 ng/uL)
BsaI 2 uL 2 uL 2 uL
DNA (500 ng)8.14 uL 18.01 uL8.83 uL
10x Cutsmart 5 uL 5 uL 5 uL
ddH2O 34.86 uL 25 uL 34.17 uL
Total 50 uL 50 uL 50 uL
50 C 2 hr
65 C 20 min

*For 2BC, we combined 8.84 uL of 33.9 ng/uL solution and 9.17 uL and 33.9 ng/uL solution to get 500 ng total.

2.2 Purification

∙ PCR purified the post-elution PCR digestion products and got the following yields:

Concentration (ng/uL)A (260/280)
2AB3.74 1.22
2BC9.81 2.54
2CA11.38 2.33

Table 1: PCR purification of BsaI digested fragments generated through PCR using post-elution primers.

∙ Yield and absorbance for the digested inserts are not very good. This might be a problem due to the PCR process, even though product was generated.

3 Bacterial Propagation

∙ Grew 7 mL each of AB, BC, CA from glycerol stock for DNA extraction tomorrow.