Revision as of 17:03, 29 April 2013 by >Djacobse
Urea Gel Quantification
Protocol 1
1. Prerun gel for at least 20 minutes at 250 Volts
2. Prepare samples, typical setup:
|
uL sample
|
uL ladder
|
uL TBE
|
uL 2X urea buffer
|
uL loaded
|
samples
|
0.5
|
0
|
5.5
|
6
|
12
|
ladder 1
|
0
|
0.5
|
5.5
|
6
|
12
|
ladder 2
|
0
|
1
|
5
|
6
|
12
|
3. Denature samples for 8 minutes at 65C
4. Snap cool using freezer box
5. Load samples onto gel and run for 20-25 minutes at 250V
6. Stain with 3 uL SYBR gold for at least 15 minutes
Protocol 2
1. Preheat TBE buffer by incubating it in 60C incubator
Microwave to do it quickly, but make sure not to get the buffer too hot (ideal: 45-60C)
2. Prerun gel in heated buffer at 250V for ~15-20 minutes
3. Prepare samples according to following table:
|
uL sample
|
uL ladder
|
uL TBE
|
uL 2X urea buffer
|
uL loaded
|
samples
|
0.5
|
0
|
5.5
|
6
|
12
|
ladder 1
|
0
|
0.5
|
5.5
|
6
|
12
|
ladder 2
|
0
|
1
|
5
|
6
|
12
|
4. Denature samples for 8 minutes at 65C
5. Load samples and run gel for 20-25 minutes at 65C
6. Stain with 3 uL SYBR gold for at least 15 minutes (rec: 20-25)