Team:Goettingen/protocol Plasmid Con

Restriction of DNA

1. Use up to 1 µg of DNA in a 20 µl reaction volume. Add 1 µl enzyme, 2 µl 10x recommended buffer and add up to 20 µl water.
2. Incubate for at least 1 hour at 37°C (or enzyme specific temperature).
3. Following the incubation, add 1.5 µl SAP (shrimp alkaline phosphatase) to plasmid backbones for 5’‑dephosphorylation and incubate again at 37°C for 10-30 minutes.
4. Stop reaction by heat-inactivation at 65°C for 5 minutes.

Ligation of DNA fragments

1. Measure the concentration of fragments which should be used for the ligation reaction.
2. Calculate the ratio between insert and backbone (1:1) using the following formula:
3. Pipette all things together. Use 2 µl of 10x T4 Ligation buffer (stored at -20°C, ATP containing) and at least 1 µl T4 ligase.
4. Incubate your reaction mix for 1 hour at room temperature or over night at 16°C.
5. Use at least 4 µl of the reaction mixture for transformation. (Previous purification of the ligation product can avoid false positive colonies!)

BP recombination reaction

Perform a BP recombination reaction between an attB-flanked DNA fragment and an attP-containing donor vector to generate an entry clone:
1. Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix:
     attB-PCR product    0.6 μl
     pDONR™ vector (supercoiled, 150 ng/μl)    0.2 μl
2. Vortex BP Clonase™ enzyme mix briefly.Add 0.2 μl to the components above and mix well by vortexing briefly twice.
3. Immediately back into -80°C!
4. Incubate reaction at 25°C for 1 hour.
5. Add 2 μl of 2 μg/μl Proteinase K solution and incubate at 37°C for 10 minutes.
6. Transform competent E. coli and select for the appropriate antibiotic-resistant entry clones.

LR recombination reaction

Perform an LR recombination reaction between an attL-containing entry clone and an attR-containing destination vector to generate an expression clone:
1. Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix:
     Entry clone (supercoiled, 100-300 ng)    0.6 μl
     Destination vector (supercoiled, 150 ng/μl)    0.2 μl
2. Vortex LR Clonase™ enzyme mix briefly. Add 0.2 μl to the components above and mix well by vortexing briefly twice.
3. Immediately back into -80°C!
4. Incubate reaction at 25°C for 1 hour.
5. Add 2 μl of 2 μg/μl Proteinase K solution and incubate at 37°C for 10 minutes.
6. Transform competent E. coli and select for the appropriate antibiotic-resistant expression clones.

SEAMLESS Cloning

1. Construct primers for the desired construct with 15 bp overhangs for homologous recombination later.
2. Amplify DNA fragments, purify them and measure concentration.
3. Calculate the amount of PCR product amount you need with the formula:
     ng of insert = (2 x bp of insert x 100 (ng vector)) / bp of vector
4. Pipette 100 ng of vector and the calculated amount of insert together. Scale down the reaction from 20 µl to the smallest volume possible.
5. Add 5 fold buffer and last the 10 fold enzyme mix.
6. Incubate for 30 min at room temperature.
7. Cool the sample on ice for not more than 5 minutes.
8. Use the whole sample for transformation.

Peptide Library Construction

Starting with GFP scaffolds (Denoted pRS 316-GFPM), random loop libraries were created. Randomized amino acids were inserted at Asp-102/Asp-103 and Glu-172/Asp-173 by using the NNK method in order to create the random loop libraries. NNK sequences in primers number 13 and 16 were used to create regions I and ӀӀӀ. After having randomized DNA sequences, these two regions were assembled with region ӀӀ which remained intact. In each of the amplification and extension steps, Pfus PCR protocol was used. For the assembly of the three regions, a modified fusion PCR was used.

Figure. Peptide Library Construction

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References

Pavoor, Tej V., Yong Ku Cho, and Eric V. Shusta. "Development of GFP-based biosensors possessing the binding properties of antibodies." Proceedings of the National Academy of Sciences 106.29 (2009): 11895-11900.