lab 208

Main purpose

• Gel purification of Cyc and Nir
• USER reaction
• PCR of Nir, His-Tag Sec, His-Tag TAT
• Miniprep re-purification with columns
• Inoculation of samples for mini 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)

Results

Conclusion

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