lab 208

Main purpose

• USER reaction to integrate Biobrick K808000 (AraBAD promoter system) in pZE21 containing fluorescence markers
• PCR

Who was in the lab

Kristian, Gosia, Julia, Henrike

Procedure

USER

USER reaction to integrate Biobrick K808000 (AraBAD promoter system) in pZE21 containing either GFP SF or RFP. Did doubles for both with 7 uL of AraBAD and 15 uL to compare. Standard procedure.

PCR

Performed PCR on lambda phage as a test of polymerase efficiency. (PHUSION polymerase test kit)

PCR on HAO and AMO with USER primer pair 17 with 3:00 on 54C and 18 with 3:00 on 59C respectively. Tried out both colonies (col) and PCR DNA template (pur):

• HAO pur
• HAO pur
• HAO col
• HAO col
• AMO pur
• AMO pur
• AMO col
• AMO col

Later on the day we made another PCR on cycAX with his-tag 5 rxn with primer pair 33 and 3:30 min extension time. This time with lower temperature:

• 2x on 50C
• 2x on 45C
• 1x neg. on 45C

Results

Gel on todays PCR

• 1kb ladder
• HAO col
• HAO col
• HAO pur
• HAO pur
• AMO col
• AMO col
• AMO pur
• AMO pur
• neg
• 1 kb ladder
• polymerase test with PHUSION
• polymerase test with PHUSION
• polymerase test with X7
• polymerase test with X7

Conclusion

The positive control was good with both cases of polymerase.

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