I290C

Meghan Novotny

Figure 1. I290C in hTAAR1_pcDNA3.1 (bases 1862-1864)

Mutagenesis Primer Design - 02/03/23

ATT --> TGT

Gel Electrophoresis

Gather desired DNA samples. Write and number the names of the DNA according to their planned order in the gel.
Obtain as many 0.6 mL tubes as you have samples and label them. To each tube, add:
- 3 µL of MBG water (Fridge A)
- 2 µL of the DNA sample (PCR Product)
- 1 µL of DNA dye (Fridge A)

Fill the electrophoresis tank with recycled 0.5X TBE (Cabinet A) until the liquid level is the same on both sides of the tank and the gel is covered
Add 5 uL of DNA Ladder to first well (box in Freezer A)
Set the pipette to ~6.5 µL to make a complete transfer of the sample. Load samples according to the written order in the notebook. Do not pierce the gel.

Slide the tank cover on with the negative prod (black) on the same side as the DNA.
- Run the gel for 45 minutes at 150 V

Lane 1: DNA 1kb Ladder

Lane 2: E1371Q_1

Lane 3: E1371Q_2

Lane 4: I290C_old DNA

Lane 5: I290C_new PCR product

25 µL reaction

12.5 µL Q5 Hi-Fi 2x Master Mix

1.25 µL of 10 µM forward primer

1.25 µL of 10 µM reverse primer

1 µL of 26.7 ng/µL template TAAR1_pGHE DNA

9 µL of MBG water

25.2 nmol --> 252 µL MBG

33.6 nmol --> 336 µL MBG

PCR

Initial denaturation: 98ºC for 30 sec

25 Cycles

98ºC 10 sec

50ºC 30 sec

72ºC 500 sec (~8 minutes)

Final extension: 72ºC ~8 minutes

Hold: 4ºC

TAAR1 I290C mRNA: 1%

wtCFTR mRNA: 1%

(GIRK1) KCNJ3 mRNA: 1%

(GIRK2) KCNJ6 mRNA: 1%

DAY 6 Experiment: 1% wtCFTR, 1% I290C TAAR1, 1% KCNJ3, 1% KCNJ6

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