Difference between revisions of "Team:UIOWA/Experiments"

Introduction

Agarose gel electrophoresis is useful for evaluating DNA quality, separating linear DNA fragments by size, and for estimating relative abundance of specific DNA fragments prior to conducting cloning procedures.

Materials

o 500 ml dedicated flask

o Agarose (depends on 1% agarose gel desired)

o 30mL 1X TAE buffer

o paper towel plug

o Gel Red (keep dark - it's light sensitive)

Procedure

• Assembling Gel Bed

1.     Place Gel Bed in between the Gel Bed Holder and lock in place by pressing the Holder Walls together and locking them using the handle.

2.     Make sure the Gel Bed Holder is stable by using the Level Tool. Adjust the Holder using the knobs until it is stable.

3.     Place Well Comb.

• Preparing the Agarose

4.     Add 30mL of 1x TAE Buffer to the flask.

5.     Measure and add agarose to the flask according to percentage of gel desired.

·       1% agarose = 0.300g agarose

6.     Plug the flask with paper towel and microwave agarose and 1x TAE buffer 1 min on High until just beginning to bubble

7.     Swirl to aid in dissolving the agarose, and continue microwaving 10 seconds at a time, until all the agarose is in solution.

8.     Discard paper plug and add 1 μl of gel red for every 10 mls of agarose (10 μl per 100 ml).....[to be efficient use 1.5 uL in 30mL]

·       Gel Red is light sensitive so do this step fast and keep the gel red covered.

9.     Swirl solution.

• Pour the Gel

10.   Slowly pour into prepared gel beds (small 7 cm square) [ use 40 mls for the 10 cm rectangular gel beds.]

11.   Remove any bubbles (or move them away from the wells) created during pouring using a pipet tip.

12.   Cover the gel from light and allow to set at room temperature. (Or store Store in bags or boxes at 4^oC in the dark and create a humidified environment by adding TAE-soaked paper towels to the bag or box.)

• Gel Electrophoresis

13.   Remove Well Comb and place the Gel Bed to the Electrophoresis Machine. The wells in the gel should be closer to the Negative Terminal.

14.   Mix 10-20 uL of DNA Sample with Loading dye (5 ul per 20 uL of sample)

·       Mix by pipetting in and out or by lightly vortexing.

15.   Carefully load the Sample DNA+Loading Dye into the well. Load 2.5 uL of DNA Ladder into a well.

·       Keep track of what each well contains.

16.   Run @ 75 V - 120 V