IGEM:MIT/2007/Notebook/2007-6-13: Difference between revisions
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| Line 9: | Line 9: | ||
*Pour LB/Amp plates | *Pour LB/Amp plates | ||
=Purifying the DNA= | =Purifying the DNA= | ||
#Denature the enzymes by exposing the samples to high heat in the Master Gradient Cycler. | #Denature the enzymes by exposing the samples to high heat in the Master Gradient Cycler. | ||
| Line 24: | Line 22: | ||
##*6 ul of LB | ##*6 ul of LB | ||
Gel Order: | |||
____ ____ ____ ____ ____ ____ ____ ____ | ____ ____ ____ ____ ____ ____ ____ ____ | ||
1V 1I 2V 2I 3V 3I undige L | 1V 1I 2V 2I 3V 3I undige L | ||
| Line 32: | Line 30: | ||
Ladders | Ladders | ||
1kb Ladder 2ul | *1kb Ladder 2ul | ||
Loading Buffer 6 ul | *Loading Buffer 6 ul | ||
H20 22ul | *H20 22ul | ||
Total: 30 ul | |||
Because 1kb ladder is 500 ng/ul | Because 1kb ladder is 500 ng/ul | ||
Undigest | Undigest | ||
5 ul DNA | *5 ul DNA | ||
6.25 ul Loading Buffer | *6.25 ul Loading Buffer | ||
19 ul H20 | *19 ul H20 | ||
Total: 30.25 | *Total: 30.25 | ||
Gel | Gel run at | ||
85 Volts | |||
Current | |||
Revision as of 07:16, 13 June 2007
Grad Advisors
- Morning: Eric
- Afternoon: L-A
LAB WORK
- Purify digests on gel (EGFP and NNX)
- Ligation overnight
- Pour LB/Amp plates
Purifying the DNA
- Denature the enzymes by exposing the samples to high heat in the Master Gradient Cycler.
- Put the samples in, close lid
- Put 65 degrees C for 15 minutes
- Put heat on and press enter
- Remember to turn off the heat when done!
- Prepare the gel
- Create a gel as usual (50 ml of Agar stuff, 5 ul of SYBR Safe DNA Stain)
- Put these ingredients together
- 25 ul of digest
- 6 ul of LB
Gel Order: ____ ____ ____ ____ ____ ____ ____ ____ 1V 1I 2V 2I 3V 3I undige L dd dd d xBa1 xBa1 xBa1 EcoR1 EcoR1
Ladders
- 1kb Ladder 2ul
- Loading Buffer 6 ul
- H20 22ul
Total: 30 ul Because 1kb ladder is 500 ng/ul
Undigest
- 5 ul DNA
- 6.25 ul Loading Buffer
- 19 ul H20
- Total: 30.25
Gel run at 85 Volts
Current
