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From 2012.igem.org

week 15: 6 august - 12 august

Office work

Lab work

New electrocompetent DH5 Alfa and Mach1 E. coli cells were prepared. Transformation efficiency was determined: DH5 Alfa: 10^8 Mach1: 10^9

TuYV

• Bricking:

Last week's minipreps were digested (EcoRI, PstI), the inserts ligated into the chloramphenicol backbone from BBa_J04450 and transformed into our electrocompetent DH5 Alfa E. coli. A PCR check gave negative results, but miniprep was performed anyway.

• Expression:

Last week's minipreps were digested (XbaI, Spe1), the inserts ligated into the promotor&RBS-containing backbone from BBa_J04500 and transformed into our electrocompetent BL21 E. coli. Results were unsatisfying at first, so the entire procedure was done again on saturday.

Written by: Wouter

PLRV

Four different PLRV CP's were transformed into E.coli DH5α electrocompetent cells. After culturing the cells, we miniprepped the plasmids. Restriction check with XbaI and SpeI enzymes confirms the presence of the PLRV CP inside the plasmid.

Hepatitis B outside modification

8 August (Kees)

• PCR step 1

Step 1 of the above described pcr scheme was done using primers Rev1 and FWGeneral for part 1.1 of the gene and FW1 with RevGeneral for part 1.2 of the gene. All steps were done using phusion polymerase, because of the low chance of mutations in the gene.

Result of step 1 on 1% agarose gel.

• PCR step 2

PCR steps 1.1 and 1.2 were purified using a PCR purification kit (Thermo). The primers used for this step were Rev2 and FW general on step 1.1 FW2 and Rev general on step 1.2

Result of step 2. The double band of step 2.2 is hard to explain, but the needed product is present.

9 August (Kees)

• PCR step 3

Template used was purified step 2.2 with primers: FW3 and Rev general

An overview of Steps 1.2-3.2. It is clear that the length increases as expected. The multiplicity of bands in the latter two steps could cause a problem in the ligating-pcr step 4

The nanodrop of step 3 yielded sufficient DNA to continue working (107.2 ng/µL). 10 August (Kees)

• PCR step 4

In this step, the amplicons of step 3 and 2.1 and mixed with the primers that anneal on the outside of the final construct. The overhang between the two parts should serve as a starting point for the amplification. So, for the PCR we used: 2 µL of step 2.1 (50 ng/µL) 1 µL of step 3 (100 ng/µL) With the primers: FW general Rev 3 (including a HIS-tag) This didn’t work out. Investigation of the primers showed a shift of 5aa in primer Rev 2. This error could inhibit the annealing and elongation of the overhang of the two parts. A new primer was ordered: Rev 2.2

11 August (Kees)

• PCR step 2 (with new Rev primer)

Step 2 was repeated using the new primer. The result was fully negative, including the positive control. Most like explanation is that one of the components was not added (FW primer, enzyme)

• Repeat PCR step 2

This time the step 2.2 did work.

• Step 4 attempt 2

The primers were used as on August 10, only the DNA has changed because of the different DNA resulting from step 2.

Because of repeats in the linker, and mainly, the k-coil there could be alternative annealing sites, causing a smear.

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