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Contents 1 Lab 208 1.1 Main purpose 1.2 Who was in the lab 1.3 Procedure 1.3.1 Gels 1.3.2 Colony PCR 1.3.3 Nanodrop 1.3.4 PCR 1.3.5 PCR with Taq hot start 1.3.6 USER reaction 1.4 Results 1.4.1 Gel on HAO 1.4.2 Gel on Nir 1.5 Conclusion

Lab 208

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Main purpose

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• Colony PCR for HAO
• USER reaction

Who was in the lab

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Kristian, Julia, Natalia, Henrike

Procedure

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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:

Temperature (oC)	Time (min)	Rounds
96	10 min	1
94	30 sec	14
60 increment of -0.5C every cycle	30 sec	14
72	5 min	14
94	30 sec	25
53	30 sec	25
72	5 min	25
10		hold

USER reaction

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

Results

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Gel on HAO

1% agarose gel

• 1kb ladder
• HAO 1
• 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

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No HAO has been obtained from the colony PCR run to check transformants.

Unspecific products from Nir PCR.

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