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lab 208

Main purpose

Who was in the lab

Kristian, Henrike, Julia

Procedure

Preparation of constructs to send for sequencing

Sec2, TAT3-2, TAT3-1a, CycAX and HAO constructs were prepared for sequencing. Sample preparation was performed following the indications given by the EZ-Seq service by Macrogen:

5uL of 100~150ng/ul plasmid + 5uL of 5uM primers.

An overview of the sample composition can be found in the following link: File:Construct for sequencing - Sheet1.pdf

The samples were sent to Macrogen for sequencing.

Gradient PCR for creation of Biobricks

Made a screening to find good conditions for the extraction of our constructs for subsequent ligation into BioBricks.

conditions:

A:

• HF buffer
• 2% DMSO
• 1uL 50mM MgCl2

B:

• HF buffer
• 3% DMSO
• 2uL 50mM MgCl2

C:

• HF buffer
• 5% DMSO
• 1uL 50mM MgCl2

D:

• HF buffer
• 3% DMSO
• 1uL 50mM MgCl2

The annealing temperature was a gradient from 48C to 63C in 12 steps.

PCR to linearize pZA21::RFP::araBAD

Used GC buffer and 5% DMSO. Made three samples with varying concentrations of 50mM MgCl2: 0,5 uL, 1uL and 2 uL.

template: pZA21::RFP::araBAD

primers: 55a, 55b

program:

PCR on one of the Hello Wolrd constructs to confirm it's intact

Performed PCR on the construct containing Sec2 to check the template is correct because our PCRs on it are not working.

template: Sec2 construct

primers: sequencing primers 4_FW_pZA21 and 5_RV_pZA21

PCR on NIR1 and NIR2 via. colony PCR for template to Morten Nørholm

Results

gels

• 1: 1 kb ladder
• 2: N1 HF
• 3: N1 HF 2% DMSO 1 mL MgCl2
• 4: N1 HF 5% DMSO
• 5: N1 HF 1M Betaine
• 6: N1 GC
• 7: N1 GC 2% DMSO
• 8: N1 GC 5% DMSO
• 9: N1 GC 5% DMSO 1 mL MgCL2
• 10: N1 GC 5% DMSO 2 mL MgCl2
• 11: N1 GC 1M Betaine
• 12: N2 HF
• 13: N2 HF 2% DMSO 1 mL MgCl2
• 14: N2 HF 5% DMSO
• 15: N2 HF 1M Betaine
• 16: N2 GC
• 17: N2 GC 2% DMSO
• 18: N2 GC 5% DMSO
• 19: 1 kb ladder

• 1 kb ladder
• N2 GC 5% DMSO 1 mL MgCl2
• N2 GC 5% DMSO 2 mL MgCl2
• N2 GC 1M Betaine
• pZA21::RFP::araBAD 0,5 uL MgCl2
• pZA21::RFP::araBAD 1 uL MgCl2
• pZA21::RFP::araBAD 2 uL MgCl2
• negative pZA21::RFP::araBAD
• check Sec2
• check Sec2 (duplicate)
• negative check Sec2
• screening A1
• screening A2
• screening A3

Conclusion

Navigate to the Previous or the Next Entry The program for Nir colony PCR works as a touchdown PCR and it have a big influence if you add DMSO or Betaine. It seems as 5% DMSO and no additional MgCl2 or 1M Betaine works best and somewhat equally good for both reactions.