IGEM:Harvard/2008/Lab Notebooks/DailyBook/Week3/Chemical and Light: Difference between revisions
No edit summary |
|||
Line 3: | Line 3: | ||
[[Image:7-7monmtg slides (compatible).ppt]] | [[Image:7-7monmtg slides (compatible).ppt]] | ||
= | =The Plan= | ||
==Transformation of Parts into S1 | |||
Goal: | |||
High/ Low Constitutive Promoter + RBS + Repressor Coding Region for cI Lambda/ TetR/ LacI + Terminator(s) + pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori | |||
==Scoreboard: Keeping track of what we have and don't have== | |||
===RBS + Repressor Coding Region=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| P56||RBS + TetR (P40+P43)||Yes (7/1)||Yes (7/2)||Yes (7/2)||Yes, in E1 (7/2)|||| | |||
|- | |||
| P57||RBS + cI lambda (P40+P44)||Yes (7/1)||Yes (7/2)||Yes (7/2)||Yes, E1 (7/2)|||| | |||
|- | |||
| ||RBS + LacI |||||||||||| | |||
|} | |||
===High/ Low Constitutive Promoters + RBS + Repressor Coding Region=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| P66||High + RBS + TetR (P56 +P38)|||||||||||| | |||
|- | |||
| P67||Low + RBS + TetR (P56 + P39)|||||||||||| | |||
|- | |||
| P68||High + RBS + cI Lambda (P57 + P38)|||||||||||| | |||
|- | |||
| P69||Low + RBS + cI Lambda (P57 + P39)|||||||||||| | |||
|- | |||
| ||High + RBS + LacI|||||||||||| | |||
|- | |||
| ||Low + RBS + LacI|||||||||||| | |||
|} | |||
===High/ Low Constitutive Promoters + RBS + Repressor Coding Region + Terminator(s)=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| P70||High + RBS + TetR + Term (P66 + )|||||||||||| | |||
|- | |||
| P71||Low + RBS + TetR + Term (P67 + P39)|||||||||||| | |||
|- | |||
| P72||High + RBS + cI Lambda + Term (P68 + P38)|||||||||||| | |||
|- | |||
| P73||Low + RBS + cI Lambda + Term (P69 + P39)|||||||||||| | |||
|- | |||
| ||High + RBS + LacI + Term|||||||||||| | |||
|- | |||
| ||Low + RBS + LacI + Term|||||||||||| | |||
|} | |||
===RBS + GFP + Term=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| P45||GFP only w/ RBS & terminator||N/A||N/A||N/A||N/A||Yes (7/1)||Yes | |||
|} | |||
===p-lambda/pTet/pLac Promoter + RBS + GFP + Term=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| P15||pTet + RBS + GFP + Term||N/A||N/A||N/A||N/A||Yes ||Yes | |||
|- | |||
| P20||pLac + RBS + GFP + Term||N/A||N/A||N/A||N/A||Yes ||Yes | |||
|- | |||
| P74||pLambda + RBS + GFP + Term (P18 + P45)|||||||||||| | |||
|} | |||
===pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| P58||pTet + RBS + GFP + Term in p15a ori (P1 + P15)||Yes (7/1)||Yes (7/2)||Yes (7/2)||Yes (7/2)|||| | |||
|- | |||
| P59||pLac + RBS + GFP + Term in p15a ori (P1 + P20)||Yes (7/1)||Yes (7/2)||Yes (7/2)||Yes (7/2)|||| | |||
|- | |||
| P75||pLambda + RBS + GFP + Term in p15a ori (P1 + P74)|||||||||||| | |||
|} | |||
===High/ Low Constitutive Promoters + RBS + Repressor Coding Region + Terminator(s) + pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori=== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| ||High + RBS + TetR + Term + pTet + RBS + GFP + Term in p15a ori (P70 + P58)|||||||||||| | |||
|- | |||
| ||Low + RBS + TetR + Term + pTet + RBS + GFP + Term in p15a ori (P71 + P58)|||||||||||| | |||
|- | |||
| ||High + RBS + cI Lambda + Term + pLambda + RBS + GFP + Term in p15a ori (P72 + P75) | |||
|- | |||
| ||Low + RBS + cI Lambda + Term + pLambda + RBS + GFP + Term in p15a ori (P73 + P75) | |||
|- | |||
| ||High + RBS + LacI + Term + pLac + RBS + GFP + Term in p15a ori (+ P59) | |||
|- | |||
| ||Low + RBS + LacI + Term + pLac + RBS + GFP + Term in p15a ori (+ P59) | |||
|} | |||
===Making a new CDF vector=== | |||
====CDF ori as an insert==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| P76||modified P13 + P1 (CDF ori + p15a vector)||P1- 7/2, modified P13- 7/3|||||||||| | |||
|} | |||
====CDF ori + Resistance Cassettes as inserts==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| P79||P76 + P48 (CDF ori on p15a vector + Cm Resistance)||P48- 7/3|||||||||| | |||
|- | |||
| P80||P76 + P49 (CDF ori on p15a vector + Amp Resistance)||P49- 7/3|||||||||| | |||
|} | |||
====New Vector w/ CDF ori + Resistance Marker==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Number''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid Description''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Digested?''' | |||
| align="center" style="background:#f0f0f0;"|'''Components Purified?''' | |||
| align="center" style="background:#f0f0f0;"|'''Ligated?''' | |||
| align="center" style="background:#f0f0f0;"|'''Transformed?''' | |||
| align="center" style="background:#f0f0f0;"|'''Miniprepped?''' | |||
| align="center" style="background:#f0f0f0;"|'''Direct from Registry?''' | |||
|- | |||
| P81||P79 + P5 (CDF ori + Cm resistance on pSB3K3 vector)|||||||||||| | |||
|- | |||
| P82||P80 + P5 (CDF ori + Amp resistance on pSB3K3 vector)|||||||||||| | |||
|} | |||
=Transformation of Parts/ Plasmids into S1= | |||
==Transformation of Lac/Tet + GFP into S1== | |||
7/3: 25 mL culture of S1 was grown for electroporation and transformation. | 7/3: 25 mL culture of S1 was grown for electroporation and transformation. |
Revision as of 17:58, 9 July 2008
Goals for Week 3
Summary Powerpoint by Amy™
File:7-7monmtg slides (compatible).ppt
The Plan
Goal: High/ Low Constitutive Promoter + RBS + Repressor Coding Region for cI Lambda/ TetR/ LacI + Terminator(s) + pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori
Scoreboard: Keeping track of what we have and don't have
RBS + Repressor Coding Region
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
P56 | RBS + TetR (P40+P43) | Yes (7/1) | Yes (7/2) | Yes (7/2) | Yes, in E1 (7/2) | ||
P57 | RBS + cI lambda (P40+P44) | Yes (7/1) | Yes (7/2) | Yes (7/2) | Yes, E1 (7/2) | ||
RBS + LacI |
High/ Low Constitutive Promoters + RBS + Repressor Coding Region
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
P66 | High + RBS + TetR (P56 +P38) | ||||||
P67 | Low + RBS + TetR (P56 + P39) | ||||||
P68 | High + RBS + cI Lambda (P57 + P38) | ||||||
P69 | Low + RBS + cI Lambda (P57 + P39) | ||||||
High + RBS + LacI | |||||||
Low + RBS + LacI |
High/ Low Constitutive Promoters + RBS + Repressor Coding Region + Terminator(s)
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
P70 | High + RBS + TetR + Term (P66 + ) | ||||||
P71 | Low + RBS + TetR + Term (P67 + P39) | ||||||
P72 | High + RBS + cI Lambda + Term (P68 + P38) | ||||||
P73 | Low + RBS + cI Lambda + Term (P69 + P39) | ||||||
High + RBS + LacI + Term | |||||||
Low + RBS + LacI + Term |
RBS + GFP + Term
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
P45 | GFP only w/ RBS & terminator | N/A | N/A | N/A | N/A | Yes (7/1) | Yes |
p-lambda/pTet/pLac Promoter + RBS + GFP + Term
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
P15 | pTet + RBS + GFP + Term | N/A | N/A | N/A | N/A | Yes | Yes |
P20 | pLac + RBS + GFP + Term | N/A | N/A | N/A | N/A | Yes | Yes |
P74 | pLambda + RBS + GFP + Term (P18 + P45) |
pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
P58 | pTet + RBS + GFP + Term in p15a ori (P1 + P15) | Yes (7/1) | Yes (7/2) | Yes (7/2) | Yes (7/2) | ||
P59 | pLac + RBS + GFP + Term in p15a ori (P1 + P20) | Yes (7/1) | Yes (7/2) | Yes (7/2) | Yes (7/2) | ||
P75 | pLambda + RBS + GFP + Term in p15a ori (P1 + P74) |
High/ Low Constitutive Promoters + RBS + Repressor Coding Region + Terminator(s) + pLambda/pTet/pLac Promoter + RBS + GFP + Term in Vector with p15a ori
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
High + RBS + TetR + Term + pTet + RBS + GFP + Term in p15a ori (P70 + P58) | |||||||
Low + RBS + TetR + Term + pTet + RBS + GFP + Term in p15a ori (P71 + P58) | |||||||
High + RBS + cI Lambda + Term + pLambda + RBS + GFP + Term in p15a ori (P72 + P75) | |||||||
Low + RBS + cI Lambda + Term + pLambda + RBS + GFP + Term in p15a ori (P73 + P75) | |||||||
High + RBS + LacI + Term + pLac + RBS + GFP + Term in p15a ori (+ P59) | |||||||
Low + RBS + LacI + Term + pLac + RBS + GFP + Term in p15a ori (+ P59) |
Making a new CDF vector
CDF ori as an insert
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
P76 | modified P13 + P1 (CDF ori + p15a vector) | P1- 7/2, modified P13- 7/3 |
CDF ori + Resistance Cassettes as inserts
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
P79 | P76 + P48 (CDF ori on p15a vector + Cm Resistance) | P48- 7/3 | |||||
P80 | P76 + P49 (CDF ori on p15a vector + Amp Resistance) | P49- 7/3 |
New Vector w/ CDF ori + Resistance Marker
Plasmid Number | Plasmid Description | Components Digested? | Components Purified? | Ligated? | Transformed? | Miniprepped? | Direct from Registry? |
P81 | P79 + P5 (CDF ori + Cm resistance on pSB3K3 vector) | ||||||
P82 | P80 + P5 (CDF ori + Amp resistance on pSB3K3 vector) |
Transformation of Parts/ Plasmids into S1
Transformation of Lac/Tet + GFP into S1
7/3: 25 mL culture of S1 was grown for electroporation and transformation.
Plasmid Name | uL DNA transformed | Plates grew? | Picked colonies? | Miniprepped? | Glycerol Stocks? |
P58B | 8uL | many small colonies, no GFP | Yes | ||
P59B | 8uL | 5 medium-sized orangish-pinkish colonies, GFP + | Yes | ||
P12 (from Christina) | 5 uL | lots of small, but pinkish colonies | Yes | ||
P27 | 2uL | 9 small/medium orangish colonies, also many very small white-ish colonies | Yes | ||
P28 | 2uL | many tiny colonies, no GFP | Yes | ||
P29 | 5uL | many tiny colonies, no Venus fluor | Yes | ||
P30 | 5uL | 2 small pinkish colonies, YFP+ | Yes | ||
P31 | 8uL | lots of very small colonies, no Venus | Yes | ||
P32 | 5uL | many (>50) medium orangish-pinkish colonies, no YFP | Yes |
Kemikale 'n' Lyte
RE digests 07/07/08
Repeated digests of P1, P18, P26, P38, P39, P45, P56, P57
P1B | P18 | P26A | P38B | P39A | P45B | P56B | P57B | |
DNA | 10 μL | 20 μL | 3 μL | 5 μL | 5 μL | 3 μL | 10 μL | 10 μL |
10X NEB Buffer | buffer 3, 2.5 μL | buffer 2, 2.5 μL | buffer 3, 2.5 μL | buffer 2, 2.5 μL | buffer 2, 2.5 μL | buffer 3, 2.5 μL | buffer 3, 2.5 μL | buffer 3, 2.5 μL |
25X BSA | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL |
REs (1 μL each) | XP | SP | XP | SP | SP | XP | XP | XP |
Water | 9.5 μL | 0 μL | 16.5 μL | 14.5 μL | 14.5 μL | 16.5 μL | 9.5 μL | 9.5 μL |
07/08: Digested pCDF with XP:
- 12.5 μL water
- 1 μL 25X BSA
- 2.5 μL 10X NEB Buffer 3
- 7 μL DNA
- 1 μL of each RE (XbaI and PstI)
07/08: All of these digested plasmids were run on a 1% low melt gel.
MXH's gel extraction protocol:
- Cut out the bands
- Freeze at –20C for at least 20'
- Spin 13000 RPM 10’
- Use supernatant for ligation
- Then do gel extraction on rest of gel (QIAGEN protocol) and combine with above
Here is a ligation protocol (used by TA and MXH):
- 2 μL 5X Dilution Buffer
- 2 μL vector
- 6 μL insert
- 10 μL 2X Rapid Ligation Buffer
- 1 μL ligase
- vortex briefly
- incubate at RT for 10'
- use 5 μL to transform 50 μL cells
Transformations/Minipreps of Parts from Registries 07/07/08
07/07: We transformed P42, P11, P51, P52, P77, P78, P17, Q01121, BBa_J06911, BBa_J06912 from both the 2007 and 2008 registries (P11 was available only from the 2008 registry). We also made a positive transformation control using pUC19 DNA that came with the TOP10 cells. We made three negative plate controls using mock transformed bacteria on Amp, Kan, and Cm plates. We used E1.
FAILED TRANSFORMATIONS
2007 -- P51 (Kan), Q01121 (Kan), P17 (Kan), P52 (Kan), P78 (Kan), P77 (Kan)
2008 -- P52 (Kan), P51 (Kan), P78 (Kan), P77 (Kan), Q01121 (Kan), P17 (Kan), P11 (Cm)
The Cm and Kan mock transformations (negative controls) also failed to grow.
Alarmingly, the E1 mock transformation (no DNA, neg control) grew on the LB Carb plates. All of the Carb plates had some colonies, so we only picked from those with significantly higher numbers than the neg control. 3 colonies each from BBa_J06911 & BBa_J06912 2007 (temp sensitive LacI system) were picked and grown.
Successful Transformations
2007: P84, P85 (BBa_J06911, BBa_J06912) -- miniprepped/glycerol stocks made
The Amp negative control colonies did not grow in liquid LB Amp. The Amp positive control did grow in liquid LB Amp (pUC19).
Nanodrop of Minipreps
Plasmid | Date | Time | ng/ul | 260/280 | 260/230 |
p59 (S1) | 7/9/2008 | 4:23 PM | 84.44 | 1.9 | 2.13 |
p30 (S1) | 7/9/2008 | 4:25 PM | 40.37 | 2.23 | 2.19 |
p84a (E1) | 7/9/2008 | 4:26 PM | 291.42 | 1.96 | 2.23 |
p84b (E1) | 7/9/2008 | 4:28 PM | 349.61 | 1.91 | 2.03 |
p84c (E1) | 7/9/2008 | 4:29 PM | 237.09 | 1.92 | 1.98 |
p85a (E1) | 7/9/2008 | 4:30 PM | 327.45 | 1.94 | 2.12 |
p85b (E1) | 7/9/2008 | 4:32 PM | 331.99 | 1.95 | 2.1 |
p18 (E) | 7/9/2008 | 4:33 PM | 135.09 | 1.97 | 1.81 |
p45 (E) | 7/9/2008 | 4:34 PM | 296.22 | 1.94 | 1.99 |
p49 (E) | 7/9/2008 | 4:35 PM | 614.33 | 1.91 | 2.16 |
Part PCRs
07/07: More CDF was PCRed (w/ BioBrick adapting primers): 45μL PCR supermix, 1μL S1P13 (435ng/μL), 1μL CDF-F primer (20μM), 1μL CDF-R primer (20μM), 2μL water. Conditions: 5min @ 94°C → 35x[45s @ 94°C → 45s @ 58.7°C → 1m45s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid
07/08: P11 and P17 from 2008 (left from punch), and P26, P38, P39, P42, P51, P52, P77, P78, Q01121 from 2007 plate were PCRed: 45μL PCR supermix, 1μL DNA, 1μL BBpfx primer (20μM), 1μL BBsfx (20μM), 2μL water. Conditions: 5min @ 94°C → 35x[45s @ 94°C → 45s @ 59°C → 1m45s @72°C] → 5min @ 72°C → ∞ @ 4°C, heated lid We're PCRing these b/c transformation for amplification has not been going smoothly.
Ligations
mtrA and p45
7/7/08 Ligated mtrA and p45 using standard protocol from ligation kit. In step 2 5uL of Vector DNA was added to make a 20uL dephosphorylation mix. In step 6, 4uL of the dephosphorylated Vector DNA and 4uL insert DNA were added.
Mutant Strand Syntheis Reaction
7/8/08 Used Stratagene QuikChange Site-Directed Mutatgenesis Kit (protocol) to create a point mutation in the pstI site in mtrA. Used 5ug of primer.
Gradient PCR
7/8/08 Used gradient PCR to PCR mtrB. Cycled 30-40°C x 10 and then 52-62°C x 30.
RE digests 07/08/08
We attempted to digest the following plasmids (per the Master Plan sent out to the iGEM google group):
- A (BBa_Q01121) with ES
- P11 with ES
- P17 with ES
- P23 with EX
- P24 with EX
- P26 with XP
- P38A with ES
- P38B with XP
- P39A with ES
- P39B with XP
- P42 with XP
- P56 with EX
- P58 with EX
- P59 with EX
- P63 with ES
- P77 with ES
- P78 with ES
These digests were run on gels and the bands of the correct size (when present) were extracted.
Gels (expected sizes of parts indicated)
3% low melt agarose, visualized using EtBr/UV | ||
---|---|---|
Lane | Contents | |
1 | 100 bp ladder | |
2 | P38A PCR product with ES: 35 (NO BAND) | |
3 | P38B PCR product with XP: 35 (NO BAND) | |
4 | P39A PCR product with ES: 35 (NO BAND) | |
5 | P39B PCR product with XP: 35 (NO BAND) | |
6 | P63 with ES: 95 (extracted) |
1.5% gel (all of PCR products)
- 100 bp ladder
- A (BBa_Q01121) with ES: 1372 (NO BAND)
- P17 with ES: 902 (NO BAND)
- P26 with XP: 961 (NO BAND)
- P42 with XP: 1128 (NO BAND)
- P77 with ES: 1307 (NO BAND)
- P78 with ES: 1310 (NO BAND)
- CDF with XP: ~900 (NO BAND)
1.5% low-melt agarose gel visualized using EtBr/UV | ||
---|---|---|
Lane | Contents | |
1 | P11 with ES: 4333 (NO BAND) | |
2 | P40 with SP: 2092 (extracted) | |
3 | P23 with EX: 2157 (extracted) | |
blank | ||
4 | P24 with EX: 2187 (extracted) | |
blank | ||
5 | P56 with EX: 2752 (extracted) | |
6 | P45A with EX: 2955 (NO BAND) | |
7 | P58 with EX: 3016 (extracted) | |
8 | P45A with EX: 2955 (NO BAND) | |
9 | P59 with EX: 3201 (extracted) | |
12 | 1 KB ladder |
Ligations 07/09/08
We ligated P59 (vector) and P63 (insert); P58 (vector) and P63 (insert); P18 (vector) and P45 (insert).
- 2 μL 5X Dilution Buffer
- 2 μL vector
- 6 μL insert
- 10 μL 2X Rapid Ligation Buffer
- 1 μL ligase
- vortex briefly
- incubate at RT for 10'
- use 5 μL to transform 50 μL competent cells
We transformed the ligated plasmids (P59+63 and P58+63 on Kan, P18+45 on Amp). We also an Amp positive control of pUC19 and two mock transformations (negative controls).