Lab 208

Main purpose

• Gel purification of Cyc and Nir
• USER reaction for making His-tag on cycAX construct
• PCR of Nir, His-Tag Sec, His-Tag TAT
• Miniprep re-purification with columns
• Inoculation of samples for midi prep

Who was in the lab

Gosia, Kristian, Natalia, Julia

Procedure

Gel purification

• Gel purification was performed according to protocol included in QIAEX, Gel Extraction Kit.

USER reaction

• USER reaction was performed according to standard protocol SOP1. We use DNA linear fragment containing pZA21 bacbone,cycAX with His-Tag sequence.

PCR

• PCR according to standard PCR protocol.

Samples names:

• 1,2 - Nir 48 (primers 48a, 48b) on template of Nir (too long fragment amplified at the beginning of work with Nir PCR)
• 3,4 - Sec His-Tag, primers 19a, 19b; template: Sec 2
• 5,6 - Tat His-Tag, primers 20a, 20b; template: TAT 2 1
• 7,8 - Tat His-Tag, primers 20a, 20b; template: TAT 3 2
• 9, 10 - Tat His-Tag, primers 20a, 20b; template: TAT 3 1a
• 11, 12, 13 - negative controls as follows for: Tat His-Tag, Nir, Sec His-Tag

Program for samples:

• 1,2,12 - Program for Nir on Eppendorf machine
• 3,4,13 - 10 cycles 58.5 C annealing, 1 min elongation and 25 cycles on 67 C annealing
• 5-11 - 10 cycles on 54.5 C annealing and 25 cycles on 64 C annealing

Purification of plasmids after ethanol purification

According to protocol attached to the DNA purification kit, QIAGEN

Inoculation

For tomorrows mini prep 5 samples were prepared (all in shaker at 37 C, last line). To 10 ml of LB with kanamycin 100 uL of inoculum was added (from previous liquid culture)

PCR on Nir fragment 2 with GC-buffer

Same procedure as yesterday's PCR just with GC-buffer instead of HF buffer.

Results

Purification gel for yesterdays PCR on on cycAX with His-tag and AMO with USER primers:

• 1kb ladder
• AMO
• AMO
• AMO
• cycAX his-tag
• cycAX his-tag
• cycAX his-tag
• 1kb ladder

Gel with Nir as single gene fragments and PAO1 from environmental lab as template (only Nir E samples):

• 1kb ladder
• Nir fragment 2, expected length = 5000
• Nir fragment 2, expected length = 5000
• Nir fragment 1, expected length = 4100
• Nir fragment 1, expected length = 4100
• Nir whole fragment with KD primers, expected length = 9100
• Nir whole fragment with KD primers, expected length = 9100
• NirN, expected length = 1700
• NirN, expected length = 1700
• NirM+S, expected length = 2300
• NirM+S, expected length = 2300
• NirG, expected length = 650
• NirG, expected length = 650
• Neg. control
• 1kb ladder

Gel on todays PCR with his-tag and verification of Nir:

• 1kb ladder
• Nir previous purified tested with primers for NirG expected length = 650
• Nir previous purified tested with primers for NirG expected length = 650
• Sec His-Tag
• Sec His-Tag
• TAT His-Tag
• TAT His-Tag
• TAT His-Tag
• TAT His-Tag
• TAT His-Tag
• TAT His-Tag
• 1kb ladder

Conclusion

We have something that looks like Nir fragment 1 and further experiments will verify that. We speculate that the high GC-content can be the reason why we have had so many PCR problems with Nir and therefore we are doing the next PCR on Nir 2 with GC-buffer.

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