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Team:Stanford/NotebookPage/9 July 2010
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Today's Goal: Digest GFP, RFP, and Terminators. Will do (GFP + T) and (RFP + T) ligations on monday. <br/><br/> | Today's Goal: Digest GFP, RFP, and Terminators. Will do (GFP + T) and (RFP + T) ligations on monday. <br/><br/> | ||
'''Part #'s:''' <br/> | '''Part #'s:''' <br/> | ||
| - | GFP: E0040 (cut at S and P) <br/> | + | *GFP: E0040 (cut at S and P) <br/> |
| - | RFP: E1010 (cut at S and P) <br/> | + | *RFP: E1010 (cut at S and P) <br/> |
| - | Term: B1006 (cut at X and P) <br/><br/> | + | *Term: B1006 (cut at X and P) <br/><br/> |
'''Recipe''' <br/> | '''Recipe''' <br/> | ||
| - | 12.0 uL DNA<br/> | + | *12.0 uL DNA<br/> |
| - | 1.0 uL each enzyme<br/> | + | *1.0 uL each enzyme<br/> |
| - | 5.0 uL NEB buffer 2<br/> | + | *5.0 uL NEB buffer 2<br/> |
| - | 5 uL BSA (10x) <br/> | + | *5 uL BSA (10x) <br/> |
| - | Sterile H20 to fill up to 50 uL<br/><br/> | + | *Sterile H20 to fill up to 50 uL<br/><br/> |
Mix and put 37º waterbath for 2 or more hours. <br/><br/> | Mix and put 37º waterbath for 2 or more hours. <br/><br/> | ||
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'''Making Gel'''<br/> | '''Making Gel'''<br/> | ||
To make 50 mL of 0.8% agarose gel, add: <br/> | To make 50 mL of 0.8% agarose gel, add: <br/> | ||
| - | .4 g agarose <br/> | + | *.4 g agarose <br/> |
| - | 50 mL TAE (1x)<br/> | + | *50 mL TAE (1x)<br/> |
| - | 10 uL EtBr<br/><br/> | + | *10 uL EtBr<br/><br/> |
'''Loading the Gel''' <br/> | '''Loading the Gel''' <br/> | ||
| - | add 10 uL loading dye (6x) to digests <br/> | + | *add 10 uL loading dye (6x) to digests <br/> |
| - | add 40 uL of dye + digests to wells <br/> | + | *add 40 uL of dye + digests to wells <br/> |
| - | don't forget to include wells with controls! (uncut plasmids) <br/> | + | *don't forget to include wells with controls! (uncut plasmids) <br/> |
| - | load 10 uL ladder <br/> | + | *load 10 uL ladder <br/> |
| - | run at 95 V for about an hour <br/> | + | *run at 95 V for about an hour <br/> |
| - | *note sizes of plasmids/inserts <br/><br/> | + | **note sizes of plasmids/inserts <br/><br/> |
| - | GFP --> pSB1A2 ( | + | GFP --> pSB1A2 (<br/> |
| - | RFP --> pSB2K3 | + | RFP --> pSB2K3<br/> |
Revision as of 19:07, 19 July 2010
[http://docs.google.com/present/edit?id=0Ae31et9w_tAhZGZyc3R2ejJfOGNnY2drMmZz&hl=en&authkey=CPHGzfIB Weekly Leader Presentation: Karina]
Karina's Notebook
Today's Goal: Digest GFP, RFP, and Terminators. Will do (GFP + T) and (RFP + T) ligations on monday.
Part #'s:
- GFP: E0040 (cut at S and P)
- RFP: E1010 (cut at S and P)
- Term: B1006 (cut at X and P)
Recipe
- 12.0 uL DNA
- 1.0 uL each enzyme
- 5.0 uL NEB buffer 2
- 5 uL BSA (10x)
- Sterile H20 to fill up to 50 uL
Mix and put 37º waterbath for 2 or more hours.
Making Gel
To make 50 mL of 0.8% agarose gel, add:
- .4 g agarose
- 50 mL TAE (1x)
- 10 uL EtBr
Loading the Gel
- add 10 uL loading dye (6x) to digests
- add 40 uL of dye + digests to wells
- don't forget to include wells with controls! (uncut plasmids)
- load 10 uL ladder
- run at 95 V for about an hour
- note sizes of plasmids/inserts
- note sizes of plasmids/inserts
GFP --> pSB1A2 (
RFP --> pSB2K3
