Biotechnology Genetic Testing

Micropipetes Liquid measurements in the metric system are made in units based on the liter where a liter is about one quart. To make these precise measurements, molecular biologists use a precision tool known as a micropipet. This tool is as basic to their lab work as a hammer is to a carpenter. Micropipets come in many models and sizes. You will be using micropipets similar to those found in the Fred Hutchinson Cancer Research Center research labs

General Guidelines Set the volume only within the range of your micropipet. Have the proper size disposable tip in place on your micropipet before immersion into any solution. Always keep the micropipet in a vertical position when there is liquid in the tip. In a horizontal position, fluid can leak back into the piston. Use your thumb to control the speed at which the plunger rises after taking up or ejecting liquid. Letting the plunger snap back damages the piston and the volume dispensed.

How to use Micropipete Set amount Put on tip Press down to first stop Put into solution Release Go to location Go down to second stop Tip??

You try on paraffin– everyone at team needs to do one! 1ul 5ul 10ul 20ul 100ul 500ul Damp table will make less curl of paraffin

General Guidelines Set the volume only within the range of your micropipet. Have the proper size disposable tip in place on your micropipet before immersion into any solution. Always keep the micropipet in a vertical position when there is liquid in the tip. In a horizontal position, fluid can leak back into the piston. Use your thumb to control the speed at which the plunger rises after taking up or ejecting liquid. Letting the plunger snap back damages the piston and the volume dispensed.

How to use Micropipete Set amount Put on tip Press down to first stop Put into solution Release Go to location Go down to second stop Tip??

Hints: Gel with wells + buffer on top Set amount  put on tip Load solution (see yesterday’s notes) Tilt pipet a little  use two hands – Ms.P grabs wrist. Break through buffer, but do not stab into Gel – sample will sink into well (ok today, much better on Thursday) Stop at 1st stop  ok if some of sample left inside pipet – do not want to get air bubbles

Practice Gels! Each person at team must pipet in 15ul of solution (any color is fine) into a well Repeat! P10: 10 + 5, 7.5 + 7.5 P20: 15

Gel Boxes: how work? 1. Charges: Uses Current to “pull” molecules Black = negative = so positive will be attracted to it Red = positive = so negative will be attracted to it 2. Size: Larger objects move slower through the gel Smaller objects move faster through the gel

Data Table Observations - Red pH - Red Observations - Black pH - Black Water + NaCl + Phenol Red Buffer + Phenol Red

Gel Boxes and Power Sources See sheet for voltage amount TURN OFF when disconnecting cords! Gel Boxes and Power Sources Add 125ml distilled water + 250ul NaCl + 1ml Phenol red – mix well – turn on to ~100v – record observations + pH at both ends (red and black) Turn off  take out tray  dump down sink  dry  put back in box Add 125 TAE buffer + 1ml phenol red – mix well – turn on to ~100v – record observations + pH at both ends (red and black) Turn off  take out tray  dump down sink  dry 

Data Table Observations - Red pH - Red Observations - Black pH - Black Water + NaCl + Phenol Red Yellow/clear color – bubbles by metal bar 5-6 Bright pink, fuchsia – bubbles by metal bar 11-9 Buffer + Phenol Red 5-7

Molecule movement – Dyes to mimic DNA Pipet in 20ul of each dye into your gel (wells are in the center) – each team member does one – bring notes Person 1 and 2, Person 3 and 4, Person 5 and 6 Person 6: Set box to 100v  run for 10 min (make sure to wait for partner box before using power x Open lid  take a picture of your gel First person that pipetted will take picture can clean up

Data Table Well number from top of box Number of dye (on top of microtude) Color of dye before Dye migrated to black or red end Color of dye after Order of distance from center (1st closest to center, 8 is furthest) 1 2 3 4 5 6 7 8

Underneath/Next to your table... Draw DNA (backbone and rungs) with the following strand (you need to come up with the complementary strand): A T C G

Post-Lab Questions

Post-Lab Questions Which dye molecules (give #) had a positive charge? Explain. Which dye molecules (give #) had a negative charge? Explain. Which dye molecule was the largest? Explain. Which dye molecule was the smallest? Explain How could scientists and doctors use Gel electrolysis to analyze someone’s DNA?