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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/> | ||
| Line 23: | Line 23: | ||
'''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/> | |
| - | GFP --> | + | GFP plasmid (pSB1A2) --> 2079 bp <br/> |
| - | RFP --> | + | GFP insert --> 720 bp<br/> |
| + | RFP plasmid (pSB2K3) --> 4425 bp <br/> | ||
| + | RFP insert --> 681 bp <br/> | ||
| + | Terminator plasmid (pSB1AK3) --> 3189 bp | ||
| + | Terminator insert --> 39 bp | ||
| + | |||
| + | '''Gel Results'''<br/> | ||
| + | [add link to gel picture here] <br/> | ||
| + | |||
| + | All bands look fine except for, there were no bands for the terminators. They were so small they ran off the gel. We are considering using PCR purification to isolate these parts. <br/><br/> | ||
| + | |||
| + | '''Gel Extraction'''<br/> | ||
| + | [http://molecool.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf Follow QIAquick Gel Extraction Kit Protocol], skip isopropanol step and elute in 50 uL H20. | ||
| + | |||
| + | {| border="1" | ||
| + | !colspan="6"|Gel Extraction | ||
| + | |- | ||
| + | |Part || Tubes (g) || Gel Extract + tube (g) || Gel Extract (g) | ||
| + | |- | ||
| + | | GFP || 1.06 || 1.11 || .05 | ||
| + | |- | ||
| + | | RFP || 1.09 || 1.22 || .13 | ||
| + | |} | ||
| + | |||
| + | |||
| + | ==Laura's Lab notebook== | ||
| + | *worked with Karina and Francisco on digestions, gel, gel extraction (see Karina's notebook for details) | ||
| + | *for digestion: Francisco set up RFP, Karina set up terminator, I set up GFP | ||
Latest revision as of 21:06, 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
GFP plasmid (pSB1A2) --> 2079 bp
GFP insert --> 720 bp
RFP plasmid (pSB2K3) --> 4425 bp
RFP insert --> 681 bp
Terminator plasmid (pSB1AK3) --> 3189 bp
Terminator insert --> 39 bp
Gel Results
[add link to gel picture here]
All bands look fine except for, there were no bands for the terminators. They were so small they ran off the gel. We are considering using PCR purification to isolate these parts.
Gel Extraction
[http://molecool.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf Follow QIAquick Gel Extraction Kit Protocol], skip isopropanol step and elute in 50 uL H20.
| Gel Extraction | |||||
|---|---|---|---|---|---|
| Part | Tubes (g) | Gel Extract + tube (g) | Gel Extract (g) | ||
| GFP | 1.06 | 1.11 | .05 | ||
| RFP | 1.09 | 1.22 | .13 | ||
Laura's Lab notebook
- worked with Karina and Francisco on digestions, gel, gel extraction (see Karina's notebook for details)
- for digestion: Francisco set up RFP, Karina set up terminator, I set up GFP
