Griffitts:DNA sequencing: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
m (→DNA Prep) |
m (→DNA Prep) |
||
| (3 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
{{Griffitts}} | <div class="noprint"> | ||
{{Template:Griffitts}} | |||
</div> | |||
==DNA Prep== | ==DNA Prep== | ||
* Make a 1:10 dilution of each of your primers | * Make a 1:10 dilution of each of your primers | ||
** 3 μL primer + 27 μL ddH<sub>2</sub>0 will usually suffice | ** 3 μL primer + 27 μL ddH<sub>2</sub>0 will usually suffice | ||
* | ** NOTE: Each sequence will require two samples—one with the upstream primer and one with the downstream primer | ||
* | * Label 1.5 mL microcentrifuge tubes | ||
* Add 1 μL of the appropriate primer to each tube | * Add 1 μL of the appropriate primer to each tube | ||
* Add | * Add DNA from a [[Griffitts:PCR_clean-up|cleaned up]] [[Griffitts:TaqPCR|PCR]] according to the table below | ||
* | * Add sterile ddH<sub>2</sub>O according to the table below | ||
* Report your sequencing reactions to Dr. Griffitts or [http://dnasc.byu.edu/ online] | * Report your sequencing reactions to Dr. Griffitts or [http://dnasc.byu.edu/ online] | ||
* Take your sequencing reactions to 690 WIDB and put them in the refrigerator | * Take your sequencing reactions to 690 WIDB and put them in the refrigerator | ||
** Make sure the liquid is at the bottom of the tube | ** NOTE: Make sure the liquid is at the bottom of the tube | ||
==Recipes== | |||
If you load 4 μL of our [[Griffitts:Stock solutions#DNA ladder|DNA ladder]], the 3 Kb band will represent 12.5 ng/μL. To determine how much DNA to use for sequencing, compare the brightness (thickness) of the 3 Kb band to your DNA band then consult the table below. To sequence 600 to 800 bases, the BYU sequencing center requires 6.67 ng/μL.[http://dnasc.byu.edu/indexProtocols.asp] | |||
{| border="1" cellpadding="5" cellspacing="0" | |||
|- | |||
! width="200" style="background:#808080;" | Brightness (compared to the | |||
3 Kb band of the DNA ladder) | |||
! width="150" style="background:#808080;" | [DNA] | |||
! width="150" style="background:#808080;" | cleaned up DNA | |||
! width="150" style="background:#808080;" | ddH<sub>2</sub>O | |||
! width="150" style="background:#808080;" | 1:10 primer | |||
! width="150" style="background:#808080;" | Total | |||
|- | |||
| < 0.5X as bright | |||
| < 6.25 ng/μL | |||
! style="background:#B8B8B8;" | redo PCR | |||
! style="background:#B8B8B8;" | redo PCR | |||
! style="background:#B8B8B8;" | redo PCR | |||
| redo PCR | |||
|- | |||
| 0.5X as bright | |||
| ~ 6.25 ng/μL | |||
! style="background:#B8B8B8;" | 9.6 μL | |||
! style="background:#B8B8B8;" | 0 μL | |||
! style="background:#B8B8B8;" | 1.0 μL | |||
| 10.6 μL | |||
|- | |||
| 1X as bright | |||
| ~ 12.5 ng/μL | |||
! style="background:#B8B8B8;" | 4.8 μL | |||
! style="background:#B8B8B8;" | 4.2 μL | |||
! style="background:#B8B8B8;" | 1.0 μL | |||
| 10.0 μL | |||
|- | |||
| 2X brighter | |||
| ~ 25 ng/μL | |||
! style="background:#B8B8B8;" | 2.4 μL | |||
! style="background:#B8B8B8;" | 6.6 μL | |||
! style="background:#B8B8B8;" | 1.0 μL | |||
| 10.0 μL | |||
|- | |||
| 3X brighter | |||
| ~ 37.5 ng/μL | |||
! style="background:#B8B8B8;" | 1.6 μL | |||
! style="background:#B8B8B8;" | 7.4 μL | |||
! style="background:#B8B8B8;" | 1.0 μL | |||
| 10.0 μL | |||
|- | |||
| 4X brighter | |||
| ~ 50 ng/μL | |||
! style="background:#B8B8B8;" | 1.2 μL | |||
! style="background:#B8B8B8;" | 7.8 μL | |||
! style="background:#B8B8B8;" | 1.0 μL | |||
| 10.0 μL | |||
|- | |||
| 5X brighter | |||
| ~ 62.5 ng/μL | |||
! style="background:#B8B8B8;" | 1.0 μL | |||
! style="background:#B8B8B8;" | 8.0 μL | |||
! style="background:#B8B8B8;" | 1.0 μL | |||
| 10.0 μL | |||
|} | |||
<br> | |||
<br> | |||
Latest revision as of 10:33, 19 January 2011
DNA Prep
- Make a 1:10 dilution of each of your primers
- 3 μL primer + 27 μL ddH20 will usually suffice
- NOTE: Each sequence will require two samples—one with the upstream primer and one with the downstream primer
- Label 1.5 mL microcentrifuge tubes
- Add 1 μL of the appropriate primer to each tube
- Add DNA from a cleaned up PCR according to the table below
- Add sterile ddH2O according to the table below
- Report your sequencing reactions to Dr. Griffitts or online
- Take your sequencing reactions to 690 WIDB and put them in the refrigerator
- NOTE: Make sure the liquid is at the bottom of the tube
Recipes
If you load 4 μL of our DNA ladder, the 3 Kb band will represent 12.5 ng/μL. To determine how much DNA to use for sequencing, compare the brightness (thickness) of the 3 Kb band to your DNA band then consult the table below. To sequence 600 to 800 bases, the BYU sequencing center requires 6.67 ng/μL.[1]
| Brightness (compared to the
3 Kb band of the DNA ladder) |
[DNA] | cleaned up DNA | ddH2O | 1:10 primer | Total |
|---|---|---|---|---|---|
| < 0.5X as bright | < 6.25 ng/μL | redo PCR | redo PCR | redo PCR | redo PCR |
| 0.5X as bright | ~ 6.25 ng/μL | 9.6 μL | 0 μL | 1.0 μL | 10.6 μL |
| 1X as bright | ~ 12.5 ng/μL | 4.8 μL | 4.2 μL | 1.0 μL | 10.0 μL |
| 2X brighter | ~ 25 ng/μL | 2.4 μL | 6.6 μL | 1.0 μL | 10.0 μL |
| 3X brighter | ~ 37.5 ng/μL | 1.6 μL | 7.4 μL | 1.0 μL | 10.0 μL |
| 4X brighter | ~ 50 ng/μL | 1.2 μL | 7.8 μL | 1.0 μL | 10.0 μL |
| 5X brighter | ~ 62.5 ng/μL | 1.0 μL | 8.0 μL | 1.0 μL | 10.0 μL |
