lab 208

Main purpose

• Colony PCR for HAO
• USER reaction

Who was in the lab

Kristian, Julia, Natalia, Henrike

Procedure

Gels

Run 1% agarose gel on colony PCR products. The samples are named:

• HAO 1
• HAO 2
• HAO 3
• HAO 4
• HAO 5
• HAO 6
• HAO 7
• HAO 8

Samples 1-5 and 8 were top10 transformant colonies. Colonies 1-5 were small (1mm wide) more condense and more difficult to collect; this may be due to the proteins expressed on the membrane that make it thicker. Colony 8 was wider (3mm wide) and more sticky. Samples 6 and 7 are normal transformant colonies.

Colony PCR

To check HAO transformants from USER cloning on the 01.08.

Nanodrop

Measured Midiprep from 03.08.. No DNA was detectable in the samples. Redo purification.

PCR

PCR for araBAD with new RV primer, SPL (synthetic promoter library) in pZA21 and reference promoter in pZA21.

samples:

• ara1, ara 2 - primers 12a + 12bn, template K808000, 57C, 1:00
• spl1, spl2 - primers 52a + 52b1, template RFP in pZA21, 54C, 2:30
• ref1, ref2 - primers 52a + 52b2, template RFP in pZA21, 54C, 2:30

PCR with Taq hot start

Used AmpliGold on:

• Nir1 colony
• Nir2 colony
• Nir1 isolated fragment
• Nir1 colony with x7 polymerase instead of Taq.

Reactions where done in duplicates with 5% DMSO in all reactions and they where incubated on the following program:

USER reaction

Performed USER reaction with cycAX plus his-tag and pZA21 after standard protocol SOP1.

Results

Gel on HAO

1% agarose gel

• 1kb ladder
• HAO 1 (from top 10 transformants)
• HAO 2
• HAO 3
• HAO 4
• HAO 5
• HAO 6
• HAO 7
• HAO 8
• 1 kb ladder

Gel on Nir

1% agarose gel on PCR products

• 1kb ladder
• Nir1 GC USER primers
• Nir1 GC USER primers
• Nir1 HF USER primers
• Nir1 HF USER primers
• Nir1 GC
• Nir1 GC
• Nir1 HF
• Nir1 HF
• Nir2 GC
• Nir2 GC
• Nir2 HF
• Nir2 HF
• Neg.
• 1 kb ladder

Conclusion

No HAO has been obtained from colony PCR.

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