Lab 208

Main purpose

• Biobrick construction → CycAX and AMO plasmid isolation.

Who was in the lab

Kristian, Julia

Procedure

Midiprep CycAX col.2, AMO col.2

We want a high yield purification. 100mL of ON cultures were pelleted. The protocol provided by QIAGEN Plasmid Kit was followed.

Colony PCR to confirm HAO insert

Used Q5 master mix and made only 25uL reaction volume because there was not enough Q5 mix left to make 50uL.

Made doubles for two different primer pairs:

• FW_1_HAO_Seq, RF_2_HAO_Seq - 61C - expected fragment length: 742bp
• FW_3_HAO_Seq, RV_3_HAO_Seq - 65C - expected fragment length: 743bp

Program:

PCR for HAO with USER endings

primers: 18a, 18b

template: HAO extraction PCR fragment

Made following reaction mix as master mix for 9 reactions:

All tubes apart from the negative control contained 1uL template.

Additives:

Program:

Ran PCR on two different programs, one touchdown (-0.5C per cycle for 14 cycles, 63C→55C, then 20 additional cycles on 55C) and one ramp (63C→55CC with 0.1C/sec in 35 cycles).

Navigate to the Previous or the Next Entry