Team:LMU-Munich/Notebook/Pathway
From 2010.igem.org
(→8-07-2010) |
(→10-04-2010) |
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(116 intermediate revisions not shown) | |||
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|style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-02-2010|10-02-2010]] | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-02-2010|10-02-2010]] | ||
|style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-03-2010|10-03-2010]] | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-03-2010|10-03-2010]] | ||
+ | |- | ||
+ | |style="text-align:center"| 40 | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-04-2010|10-04-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-05-2010|10-05-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-05-2010|10-06-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-07-2010|10-07-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-08-2010|10-08-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-09-2010|10-09-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-10-2010|10-10-2010]] | ||
+ | |- | ||
+ | |style="text-align:center"| 41 | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-11-2010|10-11-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-12-2010|10-12-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-13-2010|10-13-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-14-2010|10-14-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-15-2010|10-15-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-16-2010|10-16-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-17-2010|10-17-2010]] | ||
+ | |- | ||
+ | |style="text-align:center"| 42 | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-18-2010|10-18-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-19-2010|10-19-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-20-2010|10-20-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-21-2010|10-21-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-22-2010|10-22-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-23-2010|10-23-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-24-2010|10-24-2010]] | ||
+ | |- | ||
+ | |style="text-align:center"| 43 | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-25-2010|10-25-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-26-2010|10-26-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-27-2010|10-27-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-28-2010|10-28-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-29-2010|10-29-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-30-2010|10-30-2010]] | ||
+ | |style="text-align:center"| [[Team:LMU-Munich/Notebook/Pathway#10-31-2010|10-31-2010]] | ||
|- | |- | ||
|} | |} | ||
Line 131: | Line 167: | ||
== 8-05-2010 == | == 8-05-2010 == | ||
- | + | gDNA prep (cultures from 7-28-2010) with innuPREP Bacteria DNA kit | |
+ | |||
+ | -> additionally TE-buffer (100µl EDTA [o.5M]; 500µl Tris [1M]) was made, pH=8.0 | ||
== 8-06-2010 == | == 8-06-2010 == | ||
Line 140: | Line 178: | ||
== 8-08-2010 == | == 8-08-2010 == | ||
- | + | weekend | |
== 8-09-2010 == | == 8-09-2010 == | ||
- | + | PCR were performed as follows: | |
+ | |||
+ | mastermix: | ||
+ | ::{| | ||
+ | | MQ: 93.6µl | ||
+ | |- | ||
+ | | 10xbuffer: 12.0µl | ||
+ | |- | ||
+ | | dNTP's: 2.4µl (each 10mM) | ||
+ | |- | ||
+ | | Phusion: 1.2µl (Pfu-Promega) | ||
+ | |} | ||
+ | ::->109.2µl/6 = 18.2µl each tube | ||
+ | |||
+ | ---- | ||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! | ||
+ | ! 1 | ||
+ | ! 2 | ||
+ | ! 3 | ||
+ | ! 4 | ||
+ | ! 5 | ||
+ | ! 6 | ||
+ | |- | ||
+ | | '''primer fwd''' | ||
+ | | pduAfwd (1) | ||
+ | | pduJfwd (7) | ||
+ | | 1P1D (12) | ||
+ | | mpduDfwd (9) | ||
+ | | 5P3AD (14) | ||
+ | | 5P3AD (14) | ||
+ | |- | ||
+ | | '''primer rev''' | ||
+ | | pduJrev (8) | ||
+ | | pduUrev (2) | ||
+ | | mpduDrev (10) | ||
+ | | 3P2D (13) | ||
+ | | 9P4A (15) | ||
+ | | 9P4A (15) | ||
+ | |- | ||
+ | | '''template''' | ||
+ | | Knut | ||
+ | | Knut | ||
+ | | gDNA ''citrobacter freundii'' | ||
+ | | gDNA ''c. freundii'' | ||
+ | | gDNA ''streptomyces thioluteus'', glycerolstock | ||
+ | | gDNA ''s. thioluteus'', LB prep | ||
+ | |} | ||
+ | -> no products on gel picture | ||
== 8-10-2010 == | == 8-10-2010 == | ||
- | + | PCR were performed with pdu-template: | |
+ | |||
+ | mastermix: | ||
+ | ::{| | ||
+ | |template: 0.5µl | ||
+ | |- | ||
+ | |primer fwd/rev: 1µl | ||
+ | |- | ||
+ | | MQ: 15.88µl | ||
+ | |- | ||
+ | | 10xbuffer: 5µl | ||
+ | |- | ||
+ | |DMSO: 0.625µl | ||
+ | |- | ||
+ | | dNTP's: 0.5µl (each 10mM) | ||
+ | |- | ||
+ | | Phusion: 0.4µl (Pfu-Promega) | ||
+ | ---- | ||
+ | |- | ||
+ | |25µl | ||
+ | |} | ||
+ | ::->109.2µl/6 = 18.2µl each tube | ||
+ | |||
+ | this mix was produced for all of the six primer pairs (see 9.8.10) | ||
+ | |||
+ | ::->again no spcific product was seen on the gel picture | ||
== 8-11-2010 == | == 8-11-2010 == | ||
Line 158: | Line 270: | ||
== 8-14-2010 == | == 8-14-2010 == | ||
- | + | weekend | |
== 8-15-2010 == | == 8-15-2010 == | ||
- | + | weekend | |
== 8-16-2010 == | == 8-16-2010 == | ||
Line 167: | Line 279: | ||
== 8-17-2010 == | == 8-17-2010 == | ||
- | + | PCR mastermix | |
+ | ::{| | ||
+ | | MQ: 34.4µl | ||
+ | |- | ||
+ | | 10xbuffer: 5µl | ||
+ | |- | ||
+ | | pF: 3µl | ||
+ | |- | ||
+ | | pR: 3µl | ||
+ | |- | ||
+ | | template: 2µl | ||
+ | |- | ||
+ | | dNTP's: 2µl | ||
+ | |- | ||
+ | | Pfu(GeneON): 0.6µl | ||
+ | |} | ||
+ | ::-> 50µl | ||
+ | |||
+ | *primer combination1: 1+8 (pduA+mpduJ) | ||
+ | *primer combination2: 2+7 (mpduJ+pduU) | ||
== 8-18-2010 == | == 8-18-2010 == | ||
Line 173: | Line 304: | ||
== 8-19-2010 == | == 8-19-2010 == | ||
- | + | PCR: | |
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! charge | ||
+ | ! 1 | ||
+ | ! 2 | ||
+ | ! 3 | ||
+ | |- | ||
+ | | MQ [µl] | ||
+ | | 13.2 | ||
+ | | 11.7 | ||
+ | | 8.7 | ||
+ | |- | ||
+ | | 10xbuffer [µl] | ||
+ | | 2.5 | ||
+ | | 2.5 | ||
+ | | 2.5 | ||
+ | |- | ||
+ | | DMSO [µl] | ||
+ | | 0.625 | ||
+ | | 0.625 | ||
+ | | 0.625 | ||
+ | |- | ||
+ | | pF [µl] | ||
+ | | 3 | ||
+ | | 3 | ||
+ | | 3 | ||
+ | |- | ||
+ | | pR [µl] | ||
+ | | 3 | ||
+ | | 3 | ||
+ | | 3 | ||
+ | |- | ||
+ | | dNTP's [µl] | ||
+ | | 2 | ||
+ | | 2 | ||
+ | | 2 | ||
+ | |- | ||
+ | | template [ng/µl] | ||
+ | | 0.5 | ||
+ | | 2 | ||
+ | | 5 | ||
+ | |- | ||
+ | | Pfu [µl] | ||
+ | | 0.5 | ||
+ | | 0.5 | ||
+ | | 0.5 | ||
+ | |} | ||
+ | -> each 25µl | ||
+ | * primer combination1: 1+8 (Knut [5ng;1ng]) | ||
+ | * primer combination2: 1+6 (Knut [6ng;2ng]) | ||
+ | * primer combination3: 2+5 (Knut [7ng;3ng]) | ||
+ | * primer combination4: 2+7 (Knut [8ng;4ng]) | ||
+ | ---- | ||
+ | transformation efficiency from competent cells: 5.6x10<sup>6</sup> | ||
== 8-20-2010 == | == 8-20-2010 == | ||
- | + | PCR mastermix | |
+ | ::{| | ||
+ | | MQ: 24.75µl | ||
+ | >- | ||
+ | |DMSO: 1.25µl | ||
+ | |- | ||
+ | | 10xbuffer: 5µl | ||
+ | |- | ||
+ | | pF: 3µl | ||
+ | |- | ||
+ | | pR: 3µl | ||
+ | |- | ||
+ | | template: 10µl | ||
+ | |- | ||
+ | | dNTP's: 2µl | ||
+ | |- | ||
+ | | Pfu(GeneON): 1µl | ||
+ | |} | ||
+ | ::-> 50µl | ||
+ | |||
+ | *primer combination1: 1+6 | ||
+ | *primer combination2: 2+5 | ||
== 8-21-2010 == | == 8-21-2010 == | ||
- | + | weekend | |
== 8-22-2010 == | == 8-22-2010 == | ||
- | + | weekend | |
== 8-23-2010 == | == 8-23-2010 == | ||
Line 220: | Line 426: | ||
<font color="#009933"> Test-Transformation</font> | <font color="#009933"> Test-Transformation</font> | ||
- | in order to see whether the | + | in order to see whether the cells will work |
- done with protocoll ([[Team:LMU-Munich/Notebook/Protocols/3_Transformation|3 Transformation]]) | - done with protocoll ([[Team:LMU-Munich/Notebook/Protocols/3_Transformation|3 Transformation]]) | ||
Line 233: | Line 439: | ||
== 8-25-2010 == | == 8-25-2010 == | ||
- | + | again: | |
+ | <font color="#009933"> Preparing Competent Cells</font> | ||
+ | with Benny's protocoll & buffers | ||
+ | |||
+ | - grow the culture to an OD <sub> 600 </sub> of ~ 0,3 | ||
+ | |||
+ | -> our cells: OD <sub> 600 </sub> = 0,22 | ||
+ | |||
+ | - centrifuge for 10 min at 4°C at 3000rpm | ||
+ | |||
+ | - resuspend the pellet in buffer 1 | ||
+ | |||
+ | -> we used 16 ml | ||
+ | |||
+ | - centrifuge for 10 min at 4°C at 2500rpm | ||
+ | |||
+ | - resuspend the pellet in less buffer 1 than before | ||
+ | |||
+ | -> we used 2 ml | ||
+ | |||
+ | - then we allocated the suspension into two eppendorfs | ||
+ | |||
+ | - in eppendorf + we added buffer 2 in a ratio of 1:20 = buffer 2:buffer 1 | ||
+ | |||
+ | -> we added 50µl of buffer 2 | ||
+ | |||
+ | - incubate on ice for 10 min | ||
+ | |||
+ | - aliquote the suspension and shockfreeze it at -80°C | ||
+ | |||
+ | -> we put 50 µl in each eppendorf | ||
+ | |||
+ | |||
+ | <font color="#009933"> Test-Transformation</font> | ||
+ | in order to see whether the cells will work | ||
+ | |||
+ | - done with protocoll ([[Team:LMU-Munich/Notebook/Protocols/3_Transformation|3 Transformation]]) | ||
+ | |||
+ | -> we tested with the following DNA: pUC (10pn/µl, Amp) 1 µl was used | ||
+ | |||
+ | ::therefore we made fresh pUC (10pn/µl): 0,5 µl pUC-stock (0,5µg/µl) + 25 ml H<sub>2</sub>O) | ||
+ | |||
== 8-26-2010 == | == 8-26-2010 == | ||
- | + | joining PCR | |
+ | {| | ||
+ | | * hotstart | ||
+ | |- | ||
+ | | MQ: 11.05µl | ||
+ | |- | ||
+ | | hotstart: 25µl | ||
+ | |- | ||
+ | | MgCl: 5µl | ||
+ | |- | ||
+ | | primer forw: 3µl (2-6) | ||
+ | |- | ||
+ | | primer rev: 3µl (1-5) | ||
+ | |- | ||
+ | | template: 0.45µl (1-6) / 2.5µl (2-5) | ||
+ | |} | ||
+ | 25µl | ||
+ | ---- | ||
+ | {| | ||
+ | | * Extender | ||
+ | |- | ||
+ | | MQ: 37.75µl | ||
+ | |- | ||
+ | | Puffer10x: 5.4µl | ||
+ | |- | ||
+ | | DMSO: 1.25µl | ||
+ | |- | ||
+ | | primer forw: 2µl (1-5) | ||
+ | |- | ||
+ | | primer rev: 2µl (2-6) | ||
+ | |- | ||
+ | | template: 0.45µl (1-6) / 2.5µl (2-5) | ||
+ | |- | ||
+ | | dNTP's: 2µl | ||
+ | |- | ||
+ | | polymerase: 1µl | ||
+ | |} | ||
+ | 54µl | ||
+ | ---- | ||
+ | PCR-programm: | ||
+ | * 94°C 1' | ||
+ | ---- | ||
+ | * 94°C 30'' | ||
+ | * 39°C (hot1/ex1) / 44.2°C (hot2/ex2) / 50.5°C (hot3/ex3) / 56.7°C (hot4/ex4) / 65°C (hot5/ex5) | ||
+ | * 73°C 3' (elongationtime/cycle plus 10 sec) | ||
+ | ---- | ||
+ | * 73°C 1' | ||
+ | |||
== 8-27-2010 == | == 8-27-2010 == | ||
text | text | ||
Line 246: | Line 540: | ||
text | text | ||
== 8-31-2010 == | == 8-31-2010 == | ||
- | + | * 1: PCR- pfu (Promega) | |
+ | ::{| | ||
+ | | MQ: 24.75µl | ||
+ | |- | ||
+ | | DMSO: 1.25µl | ||
+ | |- | ||
+ | | buffer10x: 5µl | ||
+ | |- | ||
+ | | primer fwd: 3µl | ||
+ | |- | ||
+ | | primer rev: 3µl | ||
+ | |- | ||
+ | | dNTP's: 2µl | ||
+ | |- | ||
+ | | template: 10µl | ||
+ | |- | ||
+ | | polymerase pfu: 1µl | ||
+ | => 50µl | ||
+ | ---- | ||
+ | PCR-programm: | ||
+ | ::{| | ||
+ | * 1: 94°C 1' | ||
+ | ---- | ||
+ | * 2: 94°C 30" | ||
+ | * 3: 52°C 30" | ||
+ | * 4: 73°C 3' | ||
+ | * 5: repeat step 2-4 35x | ||
+ | ---- | ||
+ | * 6: 73°C 3' | ||
+ | |} | ||
+ | |} | ||
+ | |||
+ | => no fragments | ||
+ | * 2: PCR- DreamTaq Green | ||
+ | ::{| | ||
+ | | MQ: 25.5µl | ||
+ | |- | ||
+ | | DMSO: 1µl | ||
+ | |- | ||
+ | | buffer10x: 5µl | ||
+ | |- | ||
+ | | primer fwd: 3µl (#1) | ||
+ | |- | ||
+ | | primer rev: 3µl (#2) | ||
+ | |- | ||
+ | | dNTP's: 2µl | ||
+ | |- | ||
+ | | template[ng/µl]: 10µl | ||
+ | |- | ||
+ | | polymerase DreamTaq: 0.5µl | ||
+ | => 50µl | ||
+ | ---- | ||
+ | PCR-programm: | ||
+ | ::{| | ||
+ | * 1: 95°C 2' | ||
+ | ---- | ||
+ | * 2: 95°C 30" | ||
+ | * 3: 52°C 30" | ||
+ | * 4: 72°C 2'30" | ||
+ | * 5: repeat step 2-4 30x | ||
+ | ---- | ||
+ | * 6: 72°C 5' | ||
+ | |} | ||
+ | |} | ||
+ | |||
+ | => no fragments with right size | ||
+ | * 3: JoiningPCR- pdu | ||
+ | ::{| | ||
+ | | primer combinations: 1+6 & 2+5 | ||
+ | |- | ||
+ | | in the same way as PCR1&2 | ||
+ | |} | ||
+ | => no fragments | ||
+ | |||
== 9-01-2010 == | == 9-01-2010 == | ||
- | + | * 1: PCR- amplification of the pdu-operon | |
+ | primer 5+8 | ||
+ | => testing the template; 2 equal charges to check template | ||
+ | ::{| | ||
+ | | PCRmastermix(2x): 10µl | ||
+ | |- | ||
+ | | primer fwd: 1.5µl | ||
+ | |- | ||
+ | | primer rev: 1.5µl | ||
+ | |- | ||
+ | | template: 2µl / 4µl | ||
+ | |- | ||
+ | | DMSO: 0.5µl | ||
+ | |- | ||
+ | | MQ: 4.5µl / 2.5µl | ||
+ | |||
+ | each charge 20µl | ||
+ | ---- | ||
+ | PCR-programm: | ||
+ | ::{| | ||
+ | * 1: 95°C 2' | ||
+ | ---- | ||
+ | * 2: 95°C 30" | ||
+ | * 3: 50°C 30" | ||
+ | * 4: 72°C 2'30" | ||
+ | * 5: repeat step 2-4 35x | ||
+ | ---- | ||
+ | * 6: 72°C 5' | ||
+ | |} | ||
+ | |} | ||
+ | * 2: testing the plasmid via gelelectrophoresis | ||
+ | * 3: PCR- amplification of AurF (from ''streptomyces thioluteus'') | ||
+ | ::{| | ||
+ | | PCRmastermix(2x): 10µl | ||
+ | |- | ||
+ | | primer fwd: 1.5µl | ||
+ | |- | ||
+ | | primer rev: 1.5µl | ||
+ | |- | ||
+ | | template: 5µl | ||
+ | |- | ||
+ | | DMSO: 0.5µl | ||
+ | |- | ||
+ | | MQ: 1.5µl | ||
+ | => 20µl | ||
+ | ---- | ||
+ | PCR-programm: | ||
+ | ::{| | ||
+ | * 1: 95°C 2' | ||
+ | ---- | ||
+ | * 2: 95°C 30" | ||
+ | * 3: 54°C 30" | ||
+ | * 4: 72°C 2'30" | ||
+ | * 5: repeat step 2-4 35x | ||
+ | ---- | ||
+ | * 6: 72°C 5' | ||
+ | |} | ||
+ | |} | ||
+ | |||
== 9-02-2010 == | == 9-02-2010 == | ||
- | + | * PCR1- pduB->pduU | |
- | == 9- | + | primer2+5 |
- | + | {| class="wikitable" border="1" | |
+ | |- | ||
+ | ! | ||
+ | ! PCR mastermix(2x)(+Taq) | ||
+ | ! primer fwd | ||
+ | ! primer rev | ||
+ | ! template [0.5µg/ml] | ||
+ | ! MQ | ||
+ | ! DMSO | ||
+ | |- | ||
+ | | a) | ||
+ | | 10µl | ||
+ | | 1.5µl | ||
+ | | 1.5µl | ||
+ | | 4µl | ||
+ | | 2.5µl | ||
+ | | 0.5µl | ||
+ | |- | ||
+ | | b) | ||
+ | | 10µl | ||
+ | | 1.5µl | ||
+ | | 1.5µl | ||
+ | | 2µl | ||
+ | | 4.5µl | ||
+ | | 0.5µl | ||
+ | |} | ||
+ | each charge 20µl | ||
+ | ---- | ||
+ | PCR-programm: | ||
+ | ::{| | ||
+ | * 1: 95°C 2' | ||
+ | ---- | ||
+ | * 2: 95°C 30" | ||
+ | * 3: 50°C 30" | ||
+ | * 4: 72°C 2'30" | ||
+ | * 5: repeat step 2-4 35x | ||
+ | ---- | ||
+ | * 6: 72°C 5' | ||
+ | |} | ||
+ | * PCR2- amplification of pdu | ||
+ | ::{| | ||
+ | | PCRmastermix: 10µl (Taq-polymerase) | ||
+ | |- | ||
+ | | primer fwd: 1.5µl | ||
+ | |- | ||
+ | | primer rev: 1.5µl | ||
+ | |- | ||
+ | | template[ng/µl]: 4µl | ||
+ | |- | ||
+ | | MQ: 2.5µl | ||
+ | |- | ||
+ | | DMSO: 0.5µl | ||
+ | |} | ||
+ | each charge 20µl | ||
+ | ---- | ||
+ | primer combinations: | ||
+ | ::{| | ||
+ | * 1: 1+4 | ||
+ | * 2: 3+6 | ||
+ | * 3: 5+8 | ||
+ | * 4: 7+2 | ||
+ | ---- | ||
+ | PCR-programm: | ||
+ | * 1: 95°C 2' | ||
+ | ---- | ||
+ | * 2: 95°C 30" | ||
+ | * 3: 50°C 30" | ||
+ | * 4: 72°C 3' | ||
+ | * 5: repeat step 2-4 35x | ||
+ | ---- | ||
+ | * 6: 72°C 5' | ||
+ | |} | ||
+ | * PCR3- amplification of pdu | ||
+ | ::{| | ||
+ | | buffer5x: 20µl (Taq-polymerase) | ||
+ | |- | ||
+ | | primer fwd: 5µl | ||
+ | |- | ||
+ | | primer rev: 5µl | ||
+ | |- | ||
+ | | template[ng/µl]: 10µl | ||
+ | |- | ||
+ | | MQ: 51.5µl | ||
+ | |- | ||
+ | | DMSO: 2.5µl | ||
+ | |- | ||
+ | | dNTP's: 5µl | ||
+ | |- | ||
+ | | Phusion polymerase: 1µl | ||
+ | |} | ||
+ | 100µl => each charge 25µl | ||
+ | ---- | ||
+ | primer combinations: | ||
+ | ::{| | ||
+ | * 1: 1+4 | ||
+ | * 2: 3+6 | ||
+ | * 3: 5+8 | ||
+ | * 4: 7+2 | ||
+ | ---- | ||
+ | PCR-programm: | ||
+ | * 1: 98°C 1' | ||
+ | ---- | ||
+ | * 2: 98°C 30" | ||
+ | * 3: 50°C 30" | ||
+ | * 4: 72°C 2'30" | ||
+ | * 5: repeat step 2-4 35x | ||
+ | ---- | ||
+ | * 6: 72°C 5' | ||
+ | |} | ||
+ | |||
+ | == 9-03-2010 == | ||
+ | PCR- amplification of pdu | ||
+ | primer 5+8 | ||
+ | ::{| | ||
+ | | MQ: 51.5µl | ||
+ | |- | ||
+ | | buffer5x: 20µl | ||
+ | |- | ||
+ | | primer fwd: 5µl | ||
+ | |- | ||
+ | | primer rev: 5µl | ||
+ | |- | ||
+ | | dNTP's: 5µl | ||
+ | |- | ||
+ | | DMSO: 2.5µl | ||
+ | |- | ||
+ | | template: 10µl | ||
+ | |- | ||
+ | | Phusion polymerase: 1µl | ||
+ | |} | ||
+ | 100µl => 5 charges, each 20µl | ||
+ | ---- | ||
+ | PCR-programm with temperature gradient: | ||
+ | * 1: 98°C 30" | ||
+ | ---- | ||
+ | * 2: 98°C 10" | ||
+ | * 3: Tx 30" | ||
+ | * 4: 72°C 1' | ||
+ | * 5: repeat step 2-4 30x | ||
+ | ---- | ||
+ | * 6: 72°C 5' | ||
+ | |||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! charge | ||
+ | ! 1 | ||
+ | ! 2 | ||
+ | ! 3 | ||
+ | ! 4 | ||
+ | ! 5 | ||
+ | |- | ||
+ | | Tx [°C] | ||
+ | | 50 | ||
+ | | 52.5 | ||
+ | | 56.4 | ||
+ | | 61 | ||
+ | | 64.7 | ||
+ | |} | ||
+ | |||
== 9-04-2010 == | == 9-04-2010 == | ||
weekend | weekend | ||
Line 259: | Line 842: | ||
== 9-06-2010 == | == 9-06-2010 == | ||
- | + | *PCRamplification of pdu | |
+ | ::{| | ||
+ | |MQ | ||
+ | |51.5µl | ||
+ | |- | ||
+ | |buffer5x | ||
+ | |20µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |5µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |5µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |2.5µl | ||
+ | |- | ||
+ | |template | ||
+ | |10µl | ||
+ | |- | ||
+ | |Phusion polymerase | ||
+ | |1µl | ||
+ | ---- | ||
+ | |- | ||
+ | |sum | ||
+ | |100µl | ||
+ | |} | ||
+ | ---- | ||
+ | |||
+ | => 5charges, each 20µl | ||
+ | ---- | ||
+ | ::{| | ||
+ | |*primercombination 1: 1+4 | ||
+ | |- | ||
+ | |*primercombination 2: 3+6 | ||
+ | |- | ||
+ | |*primercombination 3: 5+8 | ||
+ | |- | ||
+ | |*primercombination 4: 7+2 | ||
+ | |} | ||
+ | ---- | ||
+ | PCRprogramm: | ||
+ | ::{| | ||
+ | |1:98°C 30" | ||
+ | ---- | ||
+ | |- | ||
+ | |2: 98°C 10" | ||
+ | |- | ||
+ | |3: 54°C 30" | ||
+ | |- | ||
+ | |4: 72°C 1' | ||
+ | |- | ||
+ | |5: repeat 2-4 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |6: 72°C 5' | ||
+ | |- | ||
+ | |7: 15°C break | ||
+ | |} | ||
+ | |||
+ | => no fragmentes on gel | ||
+ | |||
+ | * PCRamplification of pduD | ||
+ | ::{| | ||
+ | |PCRmastermix | ||
+ | |10µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |template | ||
+ | |4µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |2.5µl | ||
+ | ---- | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |1: 94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |2: 94°C 30" | ||
+ | |- | ||
+ | |3: 50°C 30" | ||
+ | |- | ||
+ | |4: 72°C 2' | ||
+ | |- | ||
+ | |5: repeat 2-4 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |6: 72°C 10' | ||
+ | |- | ||
+ | |7: 15°C break | ||
+ | |} | ||
+ | ---- | ||
+ | ::{| | ||
+ | |*primercombination 1: 12+10 | ||
+ | |- | ||
+ | |*primercombination 2: 9+13 | ||
+ | |} | ||
+ | |||
+ | * PCRamplification of pdu | ||
+ | ::{| | ||
+ | |MQ | ||
+ | |51.5µl | ||
+ | |- | ||
+ | |buffer5x | ||
+ | |20µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |5µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |5µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |2.5µl | ||
+ | |- | ||
+ | |template | ||
+ | |10µl | ||
+ | |- | ||
+ | |Phusion polymerase | ||
+ | |1µl | ||
+ | ---- | ||
+ | |- | ||
+ | |sum | ||
+ | |100µl | ||
+ | |} | ||
+ | ---- | ||
+ | |||
+ | => 5charges, each 20µl | ||
+ | ---- | ||
+ | ::{| | ||
+ | |*primercombination 1: 1+4 | ||
+ | |} | ||
+ | |||
== 9-07-2010 == | == 9-07-2010 == | ||
- | + | PCRamplification of pduD | |
+ | *with PCR mastermix | ||
+ | ::{| | ||
+ | |PCRmastermix | ||
+ | |50µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |7.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |7.5µl | ||
+ | |- | ||
+ | |template | ||
+ | |20µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |2.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |12.5µl | ||
+ | ---- | ||
+ | |- | ||
+ | |sum | ||
+ | |100µl | ||
+ | |} | ||
+ | ---- | ||
+ | => no product on gel | ||
+ | |||
== 9-08-2010 == | == 9-08-2010 == | ||
- | + | PCRamplification of pdu-operon | |
+ | *with PCR mastermix | ||
+ | ::{| | ||
+ | |PCRmastermix | ||
+ | |10µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |template | ||
+ | |2µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |4.5µl | ||
+ | ---- | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |} | ||
+ | |||
+ | PCR programm | ||
+ | ::{| | ||
+ | |1: 94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |2: 94°C 30" | ||
+ | |- | ||
+ | |3: 50°C 30" | ||
+ | |- | ||
+ | |4: 72°C 2' | ||
+ | |- | ||
+ | |5: repeat 2-4 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |6: 72°C 10' | ||
+ | |- | ||
+ | |7: 15°C break | ||
+ | |} | ||
+ | * with Pfu polymerase | ||
+ | ::{| | ||
+ | |MQ | ||
+ | |45µl | ||
+ | |- | ||
+ | |buffer | ||
+ | |8µ | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |6µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |6µl | ||
+ | |- | ||
+ | |template | ||
+ | |8µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |4µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |2µl | ||
+ | |- | ||
+ | |Pfu polymerase | ||
+ | |1µl | ||
+ | ---- | ||
+ | |- | ||
+ | |sum | ||
+ | |80µ | ||
+ | |} | ||
+ | =>4 charges, each 20µl | ||
+ | ---- | ||
+ | ::{| | ||
+ | |*primercombination 1: 1+4 | ||
+ | |- | ||
+ | |*primercombination 2: 3+6 | ||
+ | |- | ||
+ | |*primercombination 3: 5+8 | ||
+ | |- | ||
+ | |*primercombination 4: 7+2 | ||
+ | |} | ||
+ | ---- | ||
+ | PCRprogramm as before | ||
+ | |||
== 9-09-2010 == | == 9-09-2010 == | ||
- | + | PCR amplification of pduD from ''Citrobacter freundii'' | |
+ | |||
+ | ::{| | ||
+ | |buffer10x: 2µl | ||
+ | |- | ||
+ | |primer fwd: 1.5µl | ||
+ | |- | ||
+ | |primer rev: 1.5µl | ||
+ | |- | ||
+ | |template: 2µl | ||
+ | |- | ||
+ | |DMSO: 0.5µl | ||
+ | |- | ||
+ | |MQ: 11.25µl | ||
+ | |- | ||
+ | |dNTP's: 1µl | ||
+ | |- | ||
+ | |PCRextender polymerase: 1µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |} | ||
+ | ---- | ||
+ | * primer combination 1: #1 #4 | ||
+ | * primer combination 2: #3 #6 | ||
+ | * primer combination 3: #5 #8 | ||
+ | * primer combination 4: #7 #2 | ||
+ | ---- | ||
+ | PCR programm for charge 1&2: | ||
+ | ::{| | ||
+ | |1: 94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |2: 94°C 20" | ||
+ | |- | ||
+ | |3: 50°C 20" | ||
+ | |- | ||
+ | |4: 72°C 40" | ||
+ | |- | ||
+ | |5: go to 2; 35x | ||
+ | ---- | ||
+ | |- | ||
+ | |6: 72°C 10' | ||
+ | |} | ||
+ | PCR programm for charge 3&4: | ||
+ | ::{| | ||
+ | |1: 94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |2: 94°C 20" | ||
+ | |- | ||
+ | |3: 52°C 20" | ||
+ | |- | ||
+ | |4: 72°C 1'30" | ||
+ | |- | ||
+ | |5: go to 2; 35x | ||
+ | ---- | ||
+ | |- | ||
+ | |6: 72°C 10' | ||
+ | |} | ||
+ | PCRamplification of pduD | ||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! charge | ||
+ | ! 1 | ||
+ | ! 3 | ||
+ | ! 2 | ||
+ | ! 4 | ||
+ | |- | ||
+ | | PCRmastermix | ||
+ | | 10 | ||
+ | | 10 | ||
+ | | 10 | ||
+ | | 10 | ||
+ | |- | ||
+ | | primer fwd | ||
+ | | 1.5µl | ||
+ | | 1.5µl | ||
+ | | 1.5µl | ||
+ | | 1.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | | 1.5µl | ||
+ | | 1.5µl | ||
+ | | 1.5µl | ||
+ | | 1.5µl | ||
+ | |- | ||
+ | |template | ||
+ | |2 | ||
+ | |2 | ||
+ | |4 | ||
+ | |4 | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.5µl | ||
+ | |0.5µl | ||
+ | |0.5µl | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |4.5µl | ||
+ | |4.5µl | ||
+ | |2.5µl | ||
+ | |2.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |20µl | ||
+ | |20µl | ||
+ | |20µl | ||
+ | |} | ||
+ | ---- | ||
+ | * primer combination 1&2: #1 #4 | ||
+ | * primer combination 3&4: #3 #6 | ||
+ | ---- | ||
+ | PCR programm | ||
+ | ::{| | ||
+ | |1: 94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |2: 94°C 20" | ||
+ | |- | ||
+ | |3: 52°C 20" | ||
+ | |- | ||
+ | |4: 72°C x[t] | ||
+ | |- | ||
+ | |5: go to 2; 35x | ||
+ | ---- | ||
+ | |- | ||
+ | |6: 72°C 10' | ||
+ | |} | ||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! charge | ||
+ | ! 1 | ||
+ | ! 2 | ||
+ | ! 3 | ||
+ | ! 4 | ||
+ | |- | ||
+ | | x[t] | ||
+ | | 40" | ||
+ | | 40" | ||
+ | | 1'30" | ||
+ | | 1'30" | ||
+ | |} | ||
+ | |||
== 9-10-2010 == | == 9-10-2010 == | ||
- | + | PCR amplification of pduD from ''Citrobacter freundii'' | |
+ | |||
+ | ::{| | ||
+ | |PCRmastermix: 10µl (+Taq) | ||
+ | |- | ||
+ | |primer fwd: 1.5µl | ||
+ | |- | ||
+ | |primer rev: 1.5µl | ||
+ | |- | ||
+ | |template: 2µl | ||
+ | |- | ||
+ | |DMSO: 0.5µl | ||
+ | |- | ||
+ | |MQ: 4.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |} | ||
+ | ---- | ||
+ | * primer combination 1: #12 #15 | ||
+ | * primer combination 2: #12 #13 | ||
+ | * primer combination 3: #14 #15 | ||
+ | ---- | ||
+ | PCR programm: | ||
+ | ::{| | ||
+ | |1: 94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |2: 94°C 30" | ||
+ | |- | ||
+ | |3: 52°C 30" | ||
+ | |- | ||
+ | |4: 72°C 2' | ||
+ | |- | ||
+ | |5: go to 2; 35x | ||
+ | ---- | ||
+ | |- | ||
+ | |6: 72°C 10' | ||
+ | |- | ||
+ | |7: 15°C 5' | ||
+ | |} | ||
+ | => no result | ||
+ | ---- | ||
+ | testing transformation with Sina-Science-Services compis(250µl) and knut plasmid(10µl; 0.5µg/µl) | ||
+ | |||
+ | ::*transformation as protocol from the manufacturer... | ||
+ | ::*5µl (1:40 diluted) on CAM- and AMP-plate | ||
+ | ::* rest of them(~100µl) preculture, then 1l culture with LB (CAM/AMP) | ||
+ | |||
== 9-11-2010 == | == 9-11-2010 == | ||
weekend | weekend | ||
Line 274: | Line 1,316: | ||
== 9-13-2010 == | == 9-13-2010 == | ||
- | + | touchdown PCR of pduD | |
+ | |||
+ | ::{| | ||
+ | |1: 94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |2: 94°C 30" | ||
+ | |- | ||
+ | |3: 58°C/56°C/54°C/52°C/50°C/48° 30" | ||
+ | |- | ||
+ | |4: 72°C 2' | ||
+ | |- | ||
+ | |5: (for each temperature)repeat 2-4 2x | ||
+ | ---- | ||
+ | |- | ||
+ | |6: 94°C 30" | ||
+ | |- | ||
+ | |7: 52°C 30" | ||
+ | |- | ||
+ | |8: 72°C 2' | ||
+ | |- | ||
+ | |9: repeat 6-8 29x | ||
+ | ---- | ||
+ | |- | ||
+ | |10: 72°C 10' | ||
+ | |- | ||
+ | |11: 15°C break | ||
+ | |} | ||
+ | |||
== 9-14-2010 == | == 9-14-2010 == | ||
- | + | PCR extender | |
+ | ::{| | ||
+ | |MQ | ||
+ | |2x57µl | ||
+ | |- | ||
+ | |buffer | ||
+ | |2x10µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |2x7.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |2x7.5µl | ||
+ | |- | ||
+ | |template | ||
+ | |2x10µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |2x4.5µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |2x2.5µl | ||
+ | |- | ||
+ | |polymerase | ||
+ | |2x1µl | ||
+ | |} | ||
+ | ---- | ||
+ | 2x100µl => 4charges each 50µl | ||
+ | ---- | ||
+ | primer combination as yesterday | ||
+ | |||
== 9-15-2010 == | == 9-15-2010 == | ||
- | + | ||
+ | <font color="#009933">control-gel of fragment 2, 3 (->15ng), 4 (->20mg)</font> | ||
+ | GELFOTO REINSTELLEN | ||
+ | |||
+ | |||
+ | <font color="#009933"> joining PCR-pduOperon with fragments 3 & 4 mit PCR Extender</font> | ||
+ | |||
+ | |||
+ | :buffer: 5µl | ||
+ | :MQ: 26,5 | ||
+ | :primer: 2*2µl | ||
+ | :fragment 4: 5µl | ||
+ | :fragmet 3: 4,5 µl | ||
+ | :dNTPs: 3µl | ||
+ | :DMSO: 1,5µl | ||
+ | :PCR Extender Pol.: o,5µl | ||
+ | |||
+ | sum: 50µl | ||
+ | |||
+ | |||
+ | PCR-program: | ||
+ | |||
+ | ::{| | ||
+ | |- | ||
+ | |94°C 2min | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20sec | ||
+ | |- | ||
+ | |52°C 20sec | ||
+ | |- | ||
+ | |72°C 5min | ||
+ | |- | ||
+ | |repeat these steps 35x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10min | ||
+ | |} | ||
+ | |||
+ | GELFOTO REINSTELLEN | ||
+ | |||
== 9-16-2010 == | == 9-16-2010 == | ||
- | + | <font color="#009933"> joining PCR-pduOperon with fragments 3 & 4 mit PCR Extender, primer were added later</font> | |
+ | |||
+ | charge: same as yesterday | ||
+ | |||
+ | pcr-program: | ||
+ | |||
+ | first without primer: | ||
+ | ::{| | ||
+ | |- | ||
+ | |94°C 2min | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20sec | ||
+ | |- | ||
+ | |52°C 20sec | ||
+ | |- | ||
+ | |72°C 5min | ||
+ | |- | ||
+ | |repeat these steps 10x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10min | ||
+ | |} | ||
+ | |||
+ | then with primer: | ||
+ | ::{| | ||
+ | |- | ||
+ | |94°C 2min | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20sec | ||
+ | |- | ||
+ | |52°C 20sec | ||
+ | |- | ||
+ | |72°C 5min | ||
+ | |- | ||
+ | |repeat these steps 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10min | ||
+ | |} | ||
+ | |||
+ | GELFOTO REINSTELLEN | ||
+ | |||
+ | ->reproducable results | ||
+ | |||
+ | <font color="#009933"> control-gel and eluation of the joining-fragments</font> | ||
+ | |||
+ | GELFOTO REINSTELLEN | ||
+ | |||
+ | <font color="#009933"> purification of pdu-fragment1</font> | ||
+ | |||
+ | precipitation with EtOH + Na-Acetat (pH5,2) | ||
+ | |||
+ | precipitation of DNA-fragments <200bp | ||
+ | |||
+ | 1 vol. DNA (z.B. 100µl) | ||
+ | |||
+ | 1/10 vol. 3M Na-Acetat pH5,2 | ||
+ | |||
+ | 1µl Glycogen (optional) | ||
+ | |||
+ | 2 vol. 100% EtPH, -20°C | ||
+ | |||
+ | ::-> -20°C, 30min | ||
+ | |||
+ | ::-> max rpm, 15min | ||
+ | |||
+ | ::->discard supernatant (with pipett) | ||
+ | |||
+ | ::->"flick-spin" | ||
+ | |||
+ | ::->discard the rest of the supernatant | ||
+ | |||
+ | ::->dry at room temperature 5-10min | ||
+ | |||
+ | ::->resolve in MQ | ||
+ | |||
== 9-17-2010 == | == 9-17-2010 == | ||
- | + | <font color="#009933">joining-PCR of the pduOperon fragment1&2</font> | |
+ | |||
+ | for 100µl joining-template with PCRextender | ||
+ | ::{| | ||
+ | |- | ||
+ | |Buffer 5x | ||
+ | |5µl | ||
+ | |- | ||
+ | |H<sub>2</sub>O | ||
+ | |27.75µl | ||
+ | |- | ||
+ | |dNTPs | ||
+ | |3µl | ||
+ | |- | ||
+ | |Primer 1 | ||
+ | |2µl | ||
+ | |- | ||
+ | |Primer 6 | ||
+ | |2µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |1,5µl | ||
+ | |- | ||
+ | |template 1 | ||
+ | |1.25µl | ||
+ | |- | ||
+ | |template 2 | ||
+ | |7µl | ||
+ | |- | ||
+ | |PCRextender polymerase | ||
+ | |0,5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |50µl | ||
+ | |} | ||
+ | |||
+ | PCR programm | ||
+ | |||
+ | without primer | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 20" | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | with primer | ||
+ | |||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 20" | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">PCRamplification of pduD</font> | ||
+ | |||
+ | with PCRmastermix | ||
+ | ::{| | ||
+ | |- | ||
+ | |template | ||
+ | |2µl | ||
+ | |- | ||
+ | |MasterMix for Taq | ||
+ | |10µl | ||
+ | |- | ||
+ | |Primer *2 | ||
+ | |1.5µl *2 | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0,5µl | ||
+ | |- | ||
+ | |H<sub>2</sub>O | ||
+ | |4.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | PCR-programm | ||
+ | |||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 3' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | GELPHOTO REINSTELLEN | ||
+ | |||
== 9-18-2010 == | == 9-18-2010 == | ||
weekend | weekend | ||
Line 289: | Line 1,623: | ||
== 9-20-2010 == | == 9-20-2010 == | ||
- | + | ||
+ | |||
+ | <font color="#009933">1. PCRamplification of pduD</font> | ||
+ | with PCRextender | ||
+ | ::{| | ||
+ | |buffer | ||
+ | |5µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |2µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |2µl | ||
+ | |- | ||
+ | |template | ||
+ | |5µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |3µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |31µl | ||
+ | |- | ||
+ | |PCRextender polymerase | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |50µl | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 20" | ||
+ | |- | ||
+ | |72°C 3' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | => no result | ||
+ | |||
+ | <font color="#009933">2. purification of joining-product 1/2 from yesterday</font> | ||
+ | |||
+ | |||
+ | <font color="#009933">3. reamplification of joining-product 3/4</font> | ||
+ | |||
+ | with PCRextender | ||
+ | ::{| | ||
+ | |buffer | ||
+ | |5µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |2µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |2µl | ||
+ | |- | ||
+ | |template | ||
+ | |5µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |3µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |31µl | ||
+ | |- | ||
+ | |PCRextender polymerase | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |50µl | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
== 9-21-2010 == | == 9-21-2010 == | ||
- | + | ||
+ | <font color="#009933">test amplification of pdu-operon</font> | ||
+ | with PCRmastermix | ||
+ | ::{| | ||
+ | |PCRmastermix | ||
+ | |10µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |template (Knutplasmid) | ||
+ | |2µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |4.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 30" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">test amplification of pdu-operon</font> | ||
+ | |||
+ | with PCRextender | ||
+ | |||
+ | 5 charges: | ||
+ | ::{| | ||
+ | |buffer | ||
+ | |2µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |1µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |1µl | ||
+ | |- | ||
+ | |template | ||
+ | |2µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |2µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |11µl | ||
+ | |- | ||
+ | |PCRextender pol | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! charge | ||
+ | ! 1 | ||
+ | ! 2 | ||
+ | ! 3 | ||
+ | ! 4 | ||
+ | ! 5 | ||
+ | |- | ||
+ | | Tx[°C] | ||
+ | | 50 | ||
+ | | 51.4 | ||
+ | | 54.8 | ||
+ | | 60.2 | ||
+ | | 63.6 | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |Tx 20" | ||
+ | |- | ||
+ | |72°C 3' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">colony PCR</font> | ||
+ | for BioBricks I 712074 and E 0240 with PCRmastermix | ||
+ | |||
+ | ::{| | ||
+ | |primer fwd | ||
+ | |6µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |6µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |2µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |26µl | ||
+ | |- | ||
+ | |PCRmastermix | ||
+ | |40µl | ||
+ | |- | ||
+ | |sum | ||
+ | |80µl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 30" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 3' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | GELPHOTO REINSTELLEN | ||
+ | |||
== 9-22-2010 == | == 9-22-2010 == | ||
- | + | ||
+ | <font color="#009933">1. testamplification of pdu-operon</font> (fragment 1/2) | ||
+ | |||
+ | with PCRextender | ||
+ | ::{| | ||
+ | |buffer | ||
+ | |5µl | ||
+ | |- | ||
+ | |primer#1 | ||
+ | |2µl | ||
+ | |- | ||
+ | |primer#6 | ||
+ | |2µl | ||
+ | |- | ||
+ | |template (fragment1/2) | ||
+ | |5µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |3µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |31µl | ||
+ | |- | ||
+ | |PCRextender polymerase | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |50µl | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 20" | ||
+ | |- | ||
+ | |72°C 3' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | |||
+ | <font color="#009933">2. testamplification of pdu-operon</font> (fragment 3/4) | ||
+ | |||
+ | |||
+ | with PCRextender | ||
+ | ::{| | ||
+ | |buffer | ||
+ | |5µl | ||
+ | |- | ||
+ | |primer#2 | ||
+ | |2µl | ||
+ | |- | ||
+ | |primer#5 | ||
+ | |2µl | ||
+ | |- | ||
+ | |template | ||
+ | |20µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |3µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |16µl | ||
+ | |- | ||
+ | |PCRextender polymerase | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |50µl | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 20" | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">3. digest of BoBricks</font> | ||
+ | |||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! | ||
+ | ! enzym1 | ||
+ | ! enzym2 | ||
+ | ! buffer | ||
+ | |- | ||
+ | | I 712074 | ||
+ | | EcoRI | ||
+ | | SpeI | ||
+ | | MC | ||
+ | |- | ||
+ | | E 0240 | ||
+ | | XbaI | ||
+ | | PstI | ||
+ | | D+MC | ||
+ | |- | ||
+ | | ccdB Kan | ||
+ | | EcoRI | ||
+ | | PstI | ||
+ | | H | ||
+ | |} | ||
+ | |||
+ | {| | ||
+ | |buffer | ||
+ | |2µl | ||
+ | |- | ||
+ | |enzym1 | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |enzym2 | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |BSA | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |DNA | ||
+ | |17µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20.5µl | ||
+ | |} | ||
+ | |||
+ | => no right fragments visible | ||
+ | |||
== 9-23-2010 == | == 9-23-2010 == | ||
- | + | ||
+ | <font color="#009933">digest of BioBricks</font> | ||
+ | |||
+ | with OpenWetware-protocoll " Knight: restriction digest" | ||
+ | |||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! | ||
+ | ! Enzym1 | ||
+ | ! Enzym2 | ||
+ | ! buffer | ||
+ | ! quantity of DNA(x) | ||
+ | ! antibiotic resistance | ||
+ | |- | ||
+ | | I 712074 | ||
+ | | EcoRI | ||
+ | | SpeI | ||
+ | | MC | ||
+ | | 40µl | ||
+ | | Amp | ||
+ | |- | ||
+ | | E 0420 | ||
+ | | XbaI | ||
+ | | PstI | ||
+ | | D | ||
+ | | 40µl | ||
+ | | Kam (A/K) | ||
+ | |- | ||
+ | | ccdB tet | ||
+ | | EcoRI | ||
+ | | PstI | ||
+ | | H | ||
+ | | 10µl | ||
+ | | Tet | ||
+ | |} | ||
+ | |||
+ | ::{| | ||
+ | |buffer | ||
+ | |5µl | ||
+ | |- | ||
+ | |BSA | ||
+ | |1µl | ||
+ | |- | ||
+ | |enzym1/2 | ||
+ | |each 1µl | ||
+ | |- | ||
+ | |DNA | ||
+ | |x (look table) | ||
+ | |- | ||
+ | |MQ | ||
+ | |42-xµl | ||
+ | |- | ||
+ | |sum | ||
+ | |50µl | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">joiningPCR - pdu-operon</font> (fragment3/4) | ||
+ | with PCRextender | ||
+ | ::{| | ||
+ | |buffer | ||
+ | |5µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |2µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |2µl | ||
+ | |- | ||
+ | |fragment4 | ||
+ | |5µl | ||
+ | |- | ||
+ | |fragment3 | ||
+ | |4.5µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |3µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |26.5µl | ||
+ | |- | ||
+ | |PCRextender polymerase | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |50µl | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 20" | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |- | ||
+ | |repeat 35x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | <font color="#009933">test PCRamplification of pdu-operon</font> (fragment1/2) | ||
+ | with PCRextender | ||
+ | ::{| | ||
+ | |buffer | ||
+ | |5µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |2µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |2µl | ||
+ | |- | ||
+ | |template | ||
+ | |4µl | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |3µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |32µl | ||
+ | |- | ||
+ | |PCRextender polymerase | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |50µl | ||
+ | |} | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 20" | ||
+ | |- | ||
+ | |72°C 2' | ||
+ | |- | ||
+ | |repeat 35x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">1. amplification of lctO</font> | ||
+ | |||
+ | *with PCRextender | ||
+ | |||
+ | |||
+ | ::{| | ||
+ | |primer fwd | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |template (gDNA ''lactococcus lactus'') | ||
+ | |2µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |4.5µl | ||
+ | |- | ||
+ | |PCRmastermix | ||
+ | |10µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | => 2 charges each 10µl | ||
+ | |||
+ | *similar charge for colonyPCR | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 30" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 2' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">2. amplification of aurF</font> | ||
+ | |||
+ | :*with PCRmastermix | ||
+ | |||
+ | |||
+ | ::{| | ||
+ | |primer fwd | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |template (gDNA ''streptomyces thioluteus'') | ||
+ | |2µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |4.5µl | ||
+ | |- | ||
+ | |PCRmastermix | ||
+ | |10µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | => 2 charges each 10µl | ||
+ | |||
+ | :*similar charge for colonyPCR | ||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 30" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 2' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">3. amplification of pduD</font> | ||
+ | |||
+ | with PCRmastermix | ||
+ | |||
+ | |||
+ | ::{| | ||
+ | |primer fwd | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |template (gDNA ''citrobacter freundii'') | ||
+ | |2µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |4.5µl | ||
+ | |- | ||
+ | |PCRmastermix | ||
+ | |10µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | => 2 charges each 10µl | ||
+ | |||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 30" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 2' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">4. amplification of pduC</font> | ||
+ | |||
+ | with PCRmastermix | ||
+ | |||
+ | |||
+ | ::{| | ||
+ | |primer fwd | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |template (gDNA ''citrobacter freundii'') | ||
+ | |2µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |4.5µl | ||
+ | |- | ||
+ | |PCRmastermix | ||
+ | |10µl | ||
+ | |- | ||
+ | |sum | ||
+ | |20µl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | => 2 charges each 10µl | ||
+ | |||
+ | |||
+ | PCRprogramm | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 30" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 3' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |} | ||
+ | |||
+ | GELPHOTO REINSTELLEN | ||
+ | |||
== 9-24-2010 == | == 9-24-2010 == | ||
- | + | ||
+ | <font color="#009933">amplification of pduD</font> | ||
+ | with PCRmastermix | ||
+ | ::{| | ||
+ | |- | ||
+ | |template | ||
+ | |1µl | ||
+ | |- | ||
+ | |MasterMix | ||
+ | |5µl | ||
+ | |- | ||
+ | |Primer *2 | ||
+ | |0.75µl *2 | ||
+ | |- | ||
+ | |DMSO | ||
+ | |0.25µl | ||
+ | |- | ||
+ | |H<sub>2</sub>O | ||
+ | |2.25µl | ||
+ | |- | ||
+ | |sum | ||
+ | |10µl | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | PCRprogramm: | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 30" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 2' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">amplification of pduC</font> | ||
+ | |||
+ | same charge as before(pduD) | ||
+ | |||
+ | PCRprogramm: | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 30" | ||
+ | |- | ||
+ | |52°C 30" | ||
+ | |- | ||
+ | |72°C 3' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |} | ||
+ | |||
+ | |||
+ | |||
+ | <font color="#009933">ligation of the BioBricks</font> | ||
+ | |||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! | ||
+ | ! T4buffer (10x) | ||
+ | ! Vector(30ng/µl) | ||
+ | ! Insert1(20ng/µl) | ||
+ | ! Insert2(15ng/µl) | ||
+ | ! MQ | ||
+ | ! T4 Ligase | ||
+ | ! sum | ||
+ | |- | ||
+ | | Charge1 | ||
+ | | 2µl | ||
+ | | 2µl | ||
+ | | 6µl | ||
+ | | 8µl | ||
+ | | 1µl | ||
+ | | 1µl | ||
+ | | 20µl | ||
+ | |- | ||
+ | | Charge2 | ||
+ | | 2µl | ||
+ | | 1µl | ||
+ | | 6µl | ||
+ | | 8µl | ||
+ | | 1µl | ||
+ | | 2µl | ||
+ | | 20µl | ||
+ | |} | ||
+ | |||
+ | * 30min at roomtemperature | ||
+ | * 10min denaturation at 65°C | ||
+ | |||
+ | |||
+ | <font color="#009933">joiningPCR - pduOperon</font> fragment(3/4) | ||
+ | |||
+ | with PCRextender | ||
+ | ::{| | ||
+ | |buffer | ||
+ | |5µl | ||
+ | |- | ||
+ | |primer fwd | ||
+ | |2µl | ||
+ | |- | ||
+ | |primer rev | ||
+ | |2µl | ||
+ | |- | ||
+ | |fragment4 | ||
+ | |4.5µl(20ng/µl) | ||
+ | |- | ||
+ | |fragment3 | ||
+ | |5µl(15ng/µl) | ||
+ | |- | ||
+ | |dNTP's | ||
+ | |3µl | ||
+ | |- | ||
+ | |DMSO | ||
+ | |1.5µl | ||
+ | |- | ||
+ | |MQ | ||
+ | |26.5µl | ||
+ | |- | ||
+ | |PCRextender polymerase | ||
+ | |0.5µl | ||
+ | |- | ||
+ | |sum | ||
+ | |50µl | ||
+ | |} | ||
+ | charge 3-5 s | ||
+ | |||
+ | PCRprogramm: | ||
+ | |||
+ | without primers | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 20" | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |- | ||
+ | |repeat 10x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | edit primers | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 20" | ||
+ | |- | ||
+ | |52°C 20" | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 10' | ||
+ | |} | ||
+ | |||
+ | <font color="#009933">joiningPCR - pduOperon</font> fragment(3/4) | ||
+ | |||
+ | same as before, but editing primers after 5 cyles | ||
+ | |||
+ | |||
+ | <font color="#009933">amplification of pduD</font> | ||
+ | PCR with gradient | ||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! | ||
+ | ! PCRmastermix | ||
+ | ! template | ||
+ | ! MQ | ||
+ | ! primer fwd | ||
+ | ! primer rev | ||
+ | ! DMSO | ||
+ | ! sum | ||
+ | |- | ||
+ | | D2 | ||
+ | | 8x10µl | ||
+ | | 8x2µl | ||
+ | | 8x4.5µl | ||
+ | | 8x1.5µl | ||
+ | | 8x1.5µl | ||
+ | | 8x0.5µl | ||
+ | | each20µl | ||
+ | |- | ||
+ | | D4 | ||
+ | | 8x10µl | ||
+ | | 8x4µl | ||
+ | | 8x2.5µl | ||
+ | | 8x1.5µl | ||
+ | | 8x1.5µl | ||
+ | | 8x0.5µl | ||
+ | | each20µl | ||
+ | |} | ||
+ | |||
+ | PCRprogramm: | ||
+ | ::{| | ||
+ | |94°C 2' | ||
+ | ---- | ||
+ | |- | ||
+ | |94°C 30" | ||
+ | |- | ||
+ | |Tx°C 30" | ||
+ | |- | ||
+ | |72°C 1'40" | ||
+ | |- | ||
+ | |repeat 30x | ||
+ | ---- | ||
+ | |- | ||
+ | |72°C 5' | ||
+ | |} | ||
+ | |||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! charge | ||
+ | ! D2.1 | ||
+ | ! D2.2 | ||
+ | ! D2.3 | ||
+ | ! D2.4 | ||
+ | ! D2.5 | ||
+ | ! D2.6 | ||
+ | ! D2.7 | ||
+ | ! D2.8 | ||
+ | ! D4.1 | ||
+ | ! D4.2 | ||
+ | ! D4.3 | ||
+ | ! D4.4 | ||
+ | ! D4.5 | ||
+ | ! D4.6 | ||
+ | ! D4.7 | ||
+ | ! D4.8 | ||
+ | |- | ||
+ | | Tx | ||
+ | | 45 | ||
+ | | 47.2 | ||
+ | | 52.4 | ||
+ | | 55.2 | ||
+ | | 57.8 | ||
+ | | 60.6 | ||
+ | | 65.8 | ||
+ | | 68 | ||
+ | | 45 | ||
+ | | 47.2 | ||
+ | | 52.4 | ||
+ | | 55.2 | ||
+ | | 57.8 | ||
+ | | 60.6 | ||
+ | | 65.8 | ||
+ | | 68 | ||
+ | |} | ||
+ | |||
+ | GELPHOTO REINSTELLEN | ||
+ | |||
== 9-25-2010 == | == 9-25-2010 == | ||
weekend | weekend | ||
Line 304: | Line 2,661: | ||
== 9-27-2010 == | == 9-27-2010 == | ||
- | + | transformation of KNUT(pduOperon)into BL21 | |
+ | |||
== 9-28-2010 == | == 9-28-2010 == | ||
- | + | ||
== 9-29-2010 == | == 9-29-2010 == | ||
text | text | ||
Line 312: | Line 2,670: | ||
text | text | ||
== 10-01-2010 == | == 10-01-2010 == | ||
- | + | <font color="#009933">in vivo verification of AurF</font> | |
+ | |||
+ | ''streptomyces thioluteus'' culture in LB | ||
+ | 4-aminosalicylacid-gradient and negativ controll: | ||
+ | {| class="wikitable" border="1" | ||
+ | |- | ||
+ | ! PABA | ||
+ | ! 0.5% | ||
+ | ! 1% | ||
+ | ! 2.5% | ||
+ | ! 5% | ||
+ | ! 0% | ||
+ | |} | ||
+ | |||
+ | 1ml cultures; 0.5%=>5mg 5%=>50mg | ||
+ | |||
+ | after 2h, 37°C: | ||
+ | * 0%, 0.5%, 1% show great growth | ||
+ | * 2.5%, 5% inhibition of growth | ||
+ | |||
+ | => more 4-aminosalicylacid comes to more orange product, but also minus cellquantity | ||
+ | |||
== 10-02-2010 == | == 10-02-2010 == | ||
weekend | weekend | ||
== 10-03-2010 == | == 10-03-2010 == | ||
weekend | weekend | ||
+ | |||
+ | |||
+ | == 10-04-2010 == | ||
+ | same as on friday, but charges 25ml SOB, 50µl MgCl<sub>2</sub> solution (0.2g/ml) | ||
+ | |||
+ | == 10-05-2010 == | ||
+ | text | ||
+ | == 10-06-2010 == | ||
+ | text | ||
+ | == 10-07-2010 == | ||
+ | text | ||
+ | == 10-08-2010 == | ||
+ | text | ||
+ | == 10-09-2010 == | ||
+ | weekend | ||
+ | == 10-10-2010 == | ||
+ | weekend | ||
+ | |||
+ | == 10-11-2010 == | ||
+ | text | ||
+ | == 10-12-2010 == | ||
+ | text | ||
+ | == 10-13-2010 == | ||
+ | text | ||
+ | == 10-14-2010 == | ||
+ | text | ||
+ | == 10-15-2010 == | ||
+ | text | ||
+ | == 10-16-2010 == | ||
+ | weekend | ||
+ | == 10-17-2010 == | ||
+ | weekend | ||
+ | |||
+ | == 10-18-2010 == | ||
+ | text | ||
+ | == 10-19-2010 == | ||
+ | text | ||
+ | == 10-20-2010 == | ||
+ | text | ||
+ | == 10-21-2010 == | ||
+ | text | ||
+ | == 10-22-2010 == | ||
+ | text | ||
+ | == 10-23-2010 == | ||
+ | weekend | ||
+ | == 10-24-2010 == | ||
+ | weekend | ||
+ | |||
+ | == 10-25-2010 == | ||
+ | text | ||
+ | == 10-26-2010 == | ||
+ | text | ||
+ | == 10-27-2010 == | ||
+ | text | ||
+ | == 10-28-2010 == | ||
+ | text | ||
+ | == 10-29-2010 == | ||
+ | text | ||
+ | == 10-30-2010 == | ||
+ | weekend | ||
+ | == 10-31-2010 == | ||
+ | weekend | ||
+ | |||
{{:Team:LMU-Munich/Templates/Page Footer}} | {{:Team:LMU-Munich/Templates/Page Footer}} |
Latest revision as of 15:26, 4 October 2010
Some test text in bold
We created following tests:
Example of a table
gDNA prep (cultures from 7-28-2010) with innuPREP Bacteria DNA kit
-> additionally TE-buffer (100µl EDTA [o.5M]; 500µl Tris [1M]) was made, pH=8.0
text
weekend
weekend
PCR were performed as follows:
mastermix:
-> no products on gel picture
PCR were performed with pdu-template:
mastermix:
this mix was produced for all of the six primer pairs (see 9.8.10)
text
text
text
weekend
weekend
text
PCR mastermix
text
PCR:
-> each 25µl
transformation efficiency from competent cells: 5.6x106
PCR mastermix
>-
weekend
weekend
text
Preparing Competent Cells
with Benny's protocoll & buffers
- grow the culture to an OD 600 of ~ 0,3
-> our cells: OD 600 = 0,256
- centrifuge for 10 min at 4°C at 3000rpm
- resuspend the pellet in buffer 1
-> we used 400 µl
- centrifuge for 10 min at 4°C at 2500rpm
- resuspend the pellet in less buffer 1 than before
-> we used 200 µl
- add buffer 2 in a ratio of 1:10 = buffer 2:buffer 1
-> we added 20µl of buffer 2
- incubate on ice for 10 min
-> as we probably used too little buffer, we added another 200 µl of buffer 1 and 20µl of buffer 2
- aliquote the suspension and shockfreeze it at -80°C
-> we put 50 µl in each eppendorf
- done with protocoll (3 Transformation)
-> we tested with the following DNA:
-> as the cells are probably in a to high concentration, we thined them down with saline to an end-concentration of 10 -2
again:
Preparing Competent Cells
with Benny's protocoll & buffers
- grow the culture to an OD 600 of ~ 0,3
-> our cells: OD 600 = 0,22
- centrifuge for 10 min at 4°C at 3000rpm
- resuspend the pellet in buffer 1
-> we used 16 ml
- centrifuge for 10 min at 4°C at 2500rpm
- resuspend the pellet in less buffer 1 than before
-> we used 2 ml
- then we allocated the suspension into two eppendorfs
- in eppendorf + we added buffer 2 in a ratio of 1:20 = buffer 2:buffer 1
-> we added 50µl of buffer 2
- incubate on ice for 10 min
- aliquote the suspension and shockfreeze it at -80°C
-> we put 50 µl in each eppendorf
- done with protocoll (3 Transformation)
-> we tested with the following DNA: pUC (10pn/µl, Amp) 1 µl was used
joining PCR
25µl
54µl
PCR-programm:
text
weekend
weekend
text
=> 50µl
PCR-programm:
=> no fragments
=> 50µl
PCR-programm:
=> no fragments with right size
=> no fragments
primer 5+8
=> testing the template; 2 equal charges to check template
each charge 20µl
PCR-programm:
=> 20µl
PCR-programm:
primer2+5
each charge 20µl
PCR-programm:
each charge 20µl
primer combinations:
PCR-programm:
100µl => each charge 25µl
primer combinations:
PCR-programm:
PCR- amplification of pdu
primer 5+8
100µl => 5 charges, each 20µl
PCR-programm with temperature gradient:
weekend
weekend
=> 5charges, each 20µl
PCRprogramm:
=> no fragmentes on gel
PCRprogramm
=> 5charges, each 20µl
PCRamplification of pduD
=> no product on gel
PCRamplification of pdu-operon
PCR programm
=>4 charges, each 20µl
PCRprogramm as before
PCR amplification of pduD from Citrobacter freundii
PCR programm for charge 1&2:
PCR programm for charge 3&4:
PCRamplification of pduD
PCR programm
PCR amplification of pduD from Citrobacter freundii
PCR programm:
=> no result
testing transformation with Sina-Science-Services compis(250µl) and knut plasmid(10µl; 0.5µg/µl)
weekend
weekend
touchdown PCR of pduD
PCR extender
2x100µl => 4charges each 50µl
primer combination as yesterday
control-gel of fragment 2, 3 (->15ng), 4 (->20mg)
GELFOTO REINSTELLEN
sum: 50µl
GELFOTO REINSTELLEN
joining PCR-pduOperon with fragments 3 & 4 mit PCR Extender, primer were added later
charge: same as yesterday
pcr-program:
first without primer:
then with primer:
GELFOTO REINSTELLEN
->reproducable results
control-gel and eluation of the joining-fragments
GELFOTO REINSTELLEN
purification of pdu-fragment1
precipitation with EtOH + Na-Acetat (pH5,2)
precipitation of DNA-fragments <200bp
1 vol. DNA (z.B. 100µl)
1/10 vol. 3M Na-Acetat pH5,2
1µl Glycogen (optional)
2 vol. 100% EtPH, -20°C
joining-PCR of the pduOperon fragment1&2
for 100µl joining-template with PCRextender
PCR programm
without primer
with primer
PCRamplification of pduD
with PCRmastermix
PCR-programm
GELPHOTO REINSTELLEN
weekend
weekend
1. PCRamplification of pduD
with PCRextender
PCRprogramm
=> no result
2. purification of joining-product 1/2 from yesterday
with PCRextender
PCRprogramm
test amplification of pdu-operon
with PCRmastermix
PCRprogramm
test amplification of pdu-operon
with PCRextender
5 charges:
PCRprogramm
colony PCR
for BioBricks I 712074 and E 0240 with PCRmastermix
PCRprogramm
GELPHOTO REINSTELLEN
1. testamplification of pdu-operon (fragment 1/2)
with PCRextender
PCRprogramm
PCRprogramm
3. digest of BoBricks
=> no right fragments visible
digest of BioBricks
with OpenWetware-protocoll " Knight: restriction digest"
joiningPCR - pdu-operon (fragment3/4)
with PCRextender
PCRprogramm
test PCRamplification of pdu-operon (fragment1/2)
with PCRextender
PCRprogramm
1. amplification of lctO
=> 2 charges each 10µl
PCRprogramm
2. amplification of aurF
=> 2 charges each 10µl
PCRprogramm
3. amplification of pduD
with PCRmastermix
=> 2 charges each 10µl
4. amplification of pduC
with PCRmastermix
=> 2 charges each 10µl
GELPHOTO REINSTELLEN
amplification of pduD
with PCRmastermix
PCRprogramm:
amplification of pduC
same charge as before(pduD)
PCRprogramm:
ligation of the BioBricks
with PCRextender
charge 3-5 s
PCRprogramm:
without primers
edit primers
joiningPCR - pduOperon fragment(3/4)
same as before, but editing primers after 5 cyles
PCRprogramm:
GELPHOTO REINSTELLEN
weekend
weekend
transformation of KNUT(pduOperon)into BL21
text
text
in vivo verification of AurF
streptomyces thioluteus culture in LB
4-aminosalicylacid-gradient and negativ controll:
1ml cultures; 0.5%=>5mg 5%=>50mg
after 2h, 37°C:
=> more 4-aminosalicylacid comes to more orange product, but also minus cellquantity
weekend
weekend
same as on friday, but charges 25ml SOB, 50µl MgCl2 solution (0.2g/ml)
text
text
text
text
weekend
weekend
text
text
text
text
text
weekend
weekend
text
text
text
text
text
weekend
weekend
text
text
text
text
text
weekend
weekend
Pathway Notebook
Contents
8-02-2010
8-03-2010
8-04-2010
header 1
header 2
header 3
row 1, cell 1
row 1, cell 2
row 1, cell 3
row 2, cell 1
row 2, cell 2
row 2, cell 3
8-05-2010
8-06-2010
8-07-2010
8-08-2010
8-09-2010
MQ: 93.6µl
10xbuffer: 12.0µl
dNTP's: 2.4µl (each 10mM)
Phusion: 1.2µl (Pfu-Promega)
1
2
3
4
5
6
primer fwd
pduAfwd (1)
pduJfwd (7)
1P1D (12)
mpduDfwd (9)
5P3AD (14)
5P3AD (14)
primer rev
pduJrev (8)
pduUrev (2)
mpduDrev (10)
3P2D (13)
9P4A (15)
9P4A (15)
template
Knut
Knut
gDNA citrobacter freundii
gDNA c. freundii
gDNA streptomyces thioluteus, glycerolstock
gDNA s. thioluteus, LB prep
8-10-2010
template: 0.5µl
primer fwd/rev: 1µl
MQ: 15.88µl
10xbuffer: 5µl
DMSO: 0.625µl
dNTP's: 0.5µl (each 10mM)
Phusion: 0.4µl (Pfu-Promega)
25µl
8-11-2010
8-12-2010
8-13-2010
8-14-2010
8-15-2010
8-16-2010
8-17-2010
MQ: 34.4µl
10xbuffer: 5µl
pF: 3µl
pR: 3µl
template: 2µl
dNTP's: 2µl
Pfu(GeneON): 0.6µl
8-18-2010
8-19-2010
charge
1
2
3
MQ [µl]
13.2
11.7
8.7
10xbuffer [µl]
2.5
2.5
2.5
DMSO [µl]
0.625
0.625
0.625
pF [µl]
3
3
3
pR [µl]
3
3
3
dNTP's [µl]
2
2
2
template [ng/µl]
0.5
2
5
Pfu [µl]
0.5
0.5
0.5
8-20-2010
MQ: 24.75µl
DMSO: 1.25µl
10xbuffer: 5µl
pF: 3µl
pR: 3µl
template: 10µl
dNTP's: 2µl
Pfu(GeneON): 1µl
8-21-2010
8-22-2010
8-23-2010
8-24-2010
Test-Transformation
in order to see whether the cells will work
8-25-2010
Test-Transformation
in order to see whether the cells will work
8-26-2010
* hotstart
MQ: 11.05µl
hotstart: 25µl
MgCl: 5µl
primer forw: 3µl (2-6)
primer rev: 3µl (1-5)
template: 0.45µl (1-6) / 2.5µl (2-5)
* Extender
MQ: 37.75µl
Puffer10x: 5.4µl
DMSO: 1.25µl
primer forw: 2µl (1-5)
primer rev: 2µl (2-6)
template: 0.45µl (1-6) / 2.5µl (2-5)
dNTP's: 2µl
polymerase: 1µl
8-27-2010
8-28-2010
8-29-2010
8-30-2010
8-31-2010
MQ: 24.75µl
DMSO: 1.25µl
buffer10x: 5µl
primer fwd: 3µl
primer rev: 3µl
dNTP's: 2µl
template: 10µl
polymerase pfu: 1µl
MQ: 25.5µl
DMSO: 1µl
buffer10x: 5µl
primer fwd: 3µl (#1)
primer rev: 3µl (#2)
dNTP's: 2µl
template[ng/µl]: 10µl
polymerase DreamTaq: 0.5µl
primer combinations: 1+6 & 2+5
in the same way as PCR1&2
9-01-2010
PCRmastermix(2x): 10µl
primer fwd: 1.5µl
primer rev: 1.5µl
template: 2µl / 4µl
DMSO: 0.5µl
MQ: 4.5µl / 2.5µl
PCRmastermix(2x): 10µl
primer fwd: 1.5µl
primer rev: 1.5µl
template: 5µl
DMSO: 0.5µl
MQ: 1.5µl
9-02-2010
PCR mastermix(2x)(+Taq)
primer fwd
primer rev
template [0.5µg/ml]
MQ
DMSO
a)
10µl
1.5µl
1.5µl
4µl
2.5µl
0.5µl
b)
10µl
1.5µl
1.5µl
2µl
4.5µl
0.5µl
PCRmastermix: 10µl (Taq-polymerase)
primer fwd: 1.5µl
primer rev: 1.5µl
template[ng/µl]: 4µl
MQ: 2.5µl
DMSO: 0.5µl
buffer5x: 20µl (Taq-polymerase)
primer fwd: 5µl
primer rev: 5µl
template[ng/µl]: 10µl
MQ: 51.5µl
DMSO: 2.5µl
dNTP's: 5µl
Phusion polymerase: 1µl
9-03-2010
MQ: 51.5µl
buffer5x: 20µl
primer fwd: 5µl
primer rev: 5µl
dNTP's: 5µl
DMSO: 2.5µl
template: 10µl
Phusion polymerase: 1µl
charge
1
2
3
4
5
Tx [°C]
50
52.5
56.4
61
64.7
9-04-2010
9-05-2010
9-06-2010
MQ
51.5µl
buffer5x
20µl
primer fwd
5µl
primer rev
5µl
dNTP's
5µl
DMSO
2.5µl
template
10µl
Phusion polymerase
1µl
sum
100µl
*primercombination 1: 1+4
*primercombination 2: 3+6
*primercombination 3: 5+8
*primercombination 4: 7+2
1:98°C 30"
2: 98°C 10"
3: 54°C 30"
4: 72°C 1'
5: repeat 2-4 30x
6: 72°C 5'
7: 15°C break
PCRmastermix
10µl
primer fwd
1.5µl
primer rev
1.5µl
template
4µl
DMSO
0.5µl
MQ
2.5µl
sum
20µl
1: 94°C 2'
2: 94°C 30"
3: 50°C 30"
4: 72°C 2'
5: repeat 2-4 30x
6: 72°C 10'
7: 15°C break
*primercombination 1: 12+10
*primercombination 2: 9+13
MQ
51.5µl
buffer5x
20µl
primer fwd
5µl
primer rev
5µl
dNTP's
5µl
DMSO
2.5µl
template
10µl
Phusion polymerase
1µl
sum
100µl
*primercombination 1: 1+4
9-07-2010
PCRmastermix
50µl
primer fwd
7.5µl
primer rev
7.5µl
template
20µl
DMSO
2.5µl
MQ
12.5µl
sum
100µl
9-08-2010
PCRmastermix
10µl
primer fwd
1.5µl
primer rev
1.5µl
template
2µl
DMSO
0.5µl
MQ
4.5µl
sum
20µl
1: 94°C 2'
2: 94°C 30"
3: 50°C 30"
4: 72°C 2'
5: repeat 2-4 30x
6: 72°C 10'
7: 15°C break
MQ
45µl
buffer
8µ
primer fwd
6µl
primer rev
6µl
template
8µl
dNTP's
4µl
DMSO
2µl
Pfu polymerase
1µl
sum
80µ
*primercombination 1: 1+4
*primercombination 2: 3+6
*primercombination 3: 5+8
*primercombination 4: 7+2
9-09-2010
buffer10x: 2µl
primer fwd: 1.5µl
primer rev: 1.5µl
template: 2µl
DMSO: 0.5µl
MQ: 11.25µl
dNTP's: 1µl
PCRextender polymerase: 1µl
sum
20µl
1: 94°C 2'
2: 94°C 20"
3: 50°C 20"
4: 72°C 40"
5: go to 2; 35x
6: 72°C 10'
1: 94°C 2'
2: 94°C 20"
3: 52°C 20"
4: 72°C 1'30"
5: go to 2; 35x
6: 72°C 10'
charge
1
3
2
4
PCRmastermix
10
10
10
10
primer fwd
1.5µl
1.5µl
1.5µl
1.5µl
primer rev
1.5µl
1.5µl
1.5µl
1.5µl
template
2
2
4
4
DMSO
0.5µl
0.5µl
0.5µl
0.5µl
MQ
4.5µl
4.5µl
2.5µl
2.5µl
sum
20µl
20µl
20µl
20µl
1: 94°C 2'
2: 94°C 20"
3: 52°C 20"
4: 72°C x[t]
5: go to 2; 35x
6: 72°C 10'
charge
1
2
3
4
x[t]
40"
40"
1'30"
1'30"
9-10-2010
PCRmastermix: 10µl (+Taq)
primer fwd: 1.5µl
primer rev: 1.5µl
template: 2µl
DMSO: 0.5µl
MQ: 4.5µl
sum
20µl
1: 94°C 2'
2: 94°C 30"
3: 52°C 30"
4: 72°C 2'
5: go to 2; 35x
6: 72°C 10'
7: 15°C 5'
9-11-2010
9-12-2010
9-13-2010
1: 94°C 2'
2: 94°C 30"
3: 58°C/56°C/54°C/52°C/50°C/48° 30"
4: 72°C 2'
5: (for each temperature)repeat 2-4 2x
6: 94°C 30"
7: 52°C 30"
8: 72°C 2'
9: repeat 6-8 29x
10: 72°C 10'
11: 15°C break
9-14-2010
MQ
2x57µl
buffer
2x10µl
primer fwd
2x7.5µl
primer rev
2x7.5µl
template
2x10µl
dNTP's
2x4.5µl
DMSO
2x2.5µl
polymerase
2x1µl
9-15-2010
joining PCR-pduOperon with fragments 3 & 4 mit PCR Extender
PCR-program:
94°C 2min
94°C 20sec
52°C 20sec
72°C 5min
repeat these steps 35x
72°C 10min
9-16-2010
94°C 2min
94°C 20sec
52°C 20sec
72°C 5min
repeat these steps 10x
72°C 10min
94°C 2min
94°C 20sec
52°C 20sec
72°C 5min
repeat these steps 30x
72°C 10min
9-17-2010
Buffer 5x
5µl
H2O
27.75µl
dNTPs
3µl
Primer 1
2µl
Primer 6
2µl
DMSO
1,5µl
template 1
1.25µl
template 2
7µl
PCRextender polymerase
0,5µl
sum
50µl
94°C 2'
94°C 20"
52°C 20"
72°C 5'
repeat 30x
72°C 10'
94°C 2'
94°C 20"
52°C 20"
72°C 5'
repeat 30x
72°C 10'
template
2µl
MasterMix for Taq
10µl
Primer *2
1.5µl *2
DMSO
0,5µl
H2O
4.5µl
sum
20µl
94°C 2'
94°C 20"
52°C 30"
72°C 3'
repeat 30x
72°C 10'
9-18-2010
9-19-2010
9-20-2010
buffer
5µl
primer fwd
2µl
primer rev
2µl
template
5µl
dNTP's
3µl
DMSO
1.5µl
MQ
31µl
PCRextender polymerase
0.5µl
sum
50µl
94°C 2'
94°C 20"
52°C 20"
72°C 3'
repeat 30x
72°C 10'
3. reamplification of joining-product 3/4
buffer
5µl
primer fwd
2µl
primer rev
2µl
template
5µl
dNTP's
3µl
DMSO
1.5µl
MQ
31µl
PCRextender polymerase
0.5µl
sum
50µl
94°C 2'
94°C 20"
52°C 30"
72°C 5'
repeat 30x
72°C 10'
9-21-2010
PCRmastermix
10µl
primer fwd
1.5µl
primer rev
1.5µl
template (Knutplasmid)
2µl
DMSO
0.5µl
MQ
4.5µl
sum
20µl
94°C 2'
94°C 30"
52°C 30"
72°C 5'
repeat 30x
72°C 10'
buffer
2µl
primer fwd
1µl
primer rev
1µl
template
2µl
dNTP's
2µl
DMSO
0.5µl
MQ
11µl
PCRextender pol
0.5µl
sum
20µl
charge
1
2
3
4
5
Tx[°C]
50
51.4
54.8
60.2
63.6
94°C 2'
94°C 20"
Tx 20"
72°C 3'
repeat 30x
72°C 10'
primer fwd
6µl
primer rev
6µl
DMSO
2µl
MQ
26µl
PCRmastermix
40µl
sum
80µl
94°C 2'
94°C 30"
52°C 30"
72°C 3'
repeat 30x
72°C 10'
9-22-2010
buffer
5µl
primer#1
2µl
primer#6
2µl
template (fragment1/2)
5µl
dNTP's
3µl
DMSO
1.5µl
MQ
31µl
PCRextender polymerase
0.5µl
sum
50µl
94°C 2'
94°C 20"
52°C 20"
72°C 3'
repeat 30x
72°C 10'
2. testamplification of pdu-operon (fragment 3/4)
with PCRextender
buffer
5µl
primer#2
2µl
primer#5
2µl
template
20µl
dNTP's
3µl
DMSO
1.5µl
MQ
16µl
PCRextender polymerase
0.5µl
sum
50µl
94°C 2'
94°C 20"
52°C 20"
72°C 5'
repeat 30x
72°C 10'
enzym1
enzym2
buffer
I 712074
EcoRI
SpeI
MC
E 0240
XbaI
PstI
D+MC
ccdB Kan
EcoRI
PstI
H
buffer
2µl
enzym1
0.5µl
enzym2
0.5µl
BSA
0.5µl
DNA
17µl
sum
20.5µl
9-23-2010
Enzym1
Enzym2
buffer
quantity of DNA(x)
antibiotic resistance
I 712074
EcoRI
SpeI
MC
40µl
Amp
E 0420
XbaI
PstI
D
40µl
Kam (A/K)
ccdB tet
EcoRI
PstI
H
10µl
Tet
buffer
5µl
BSA
1µl
enzym1/2
each 1µl
DNA
x (look table)
MQ
42-xµl
sum
50µl
buffer
5µl
primer fwd
2µl
primer rev
2µl
fragment4
5µl
fragment3
4.5µl
dNTP's
3µl
DMSO
1.5µl
MQ
26.5µl
PCRextender polymerase
0.5µl
sum
50µl
94°C 2'
94°C 20"
52°C 20"
72°C 5'
repeat 35x
72°C 10'
buffer
5µl
primer fwd
2µl
primer rev
2µl
template
4µl
dNTP's
3µl
DMSO
1.5µl
MQ
32µl
PCRextender polymerase
0.5µl
sum
50µl
94°C 2'
94°C 20"
52°C 20"
72°C 2'
repeat 35x
72°C 10'
primer fwd
1.5µl
primer rev
1.5µl
template (gDNA lactococcus lactus)
2µl
DMSO
0.5µl
MQ
4.5µl
PCRmastermix
10µl
sum
20µl
94°C 2'
94°C 30"
52°C 30"
72°C 2'
repeat 30x
72°C 5'
primer fwd
1.5µl
primer rev
1.5µl
template (gDNA streptomyces thioluteus)
2µl
DMSO
0.5µl
MQ
4.5µl
PCRmastermix
10µl
sum
20µl
94°C 2'
94°C 30"
52°C 30"
72°C 2'
repeat 30x
72°C 5'
primer fwd
1.5µl
primer rev
1.5µl
template (gDNA citrobacter freundii)
2µl
DMSO
0.5µl
MQ
4.5µl
PCRmastermix
10µl
sum
20µl
PCRprogramm
94°C 2'
94°C 30"
52°C 30"
72°C 2'
repeat 30x
72°C 5'
primer fwd
1.5µl
primer rev
1.5µl
template (gDNA citrobacter freundii)
2µl
DMSO
0.5µl
MQ
4.5µl
PCRmastermix
10µl
sum
20µl
PCRprogramm
94°C 2'
94°C 30"
52°C 30"
72°C 3'
repeat 30x
72°C 5'
9-24-2010
template
1µl
MasterMix
5µl
Primer *2
0.75µl *2
DMSO
0.25µl
H2O
2.25µl
sum
10µl
94°C 2'
94°C 30"
52°C 30"
72°C 2'
repeat 30x
72°C 5'
94°C 2'
94°C 30"
52°C 30"
72°C 3'
repeat 30x
72°C 5'
T4buffer (10x)
Vector(30ng/µl)
Insert1(20ng/µl)
Insert2(15ng/µl)
MQ
T4 Ligase
sum
Charge1
2µl
2µl
6µl
8µl
1µl
1µl
20µl
Charge2
2µl
1µl
6µl
8µl
1µl
2µl
20µl
joiningPCR - pduOperon fragment(3/4)
buffer
5µl
primer fwd
2µl
primer rev
2µl
fragment4
4.5µl(20ng/µl)
fragment3
5µl(15ng/µl)
dNTP's
3µl
DMSO
1.5µl
MQ
26.5µl
PCRextender polymerase
0.5µl
sum
50µl
94°C 2'
94°C 20"
52°C 20"
72°C 5'
repeat 10x
72°C 10'
94°C 2'
94°C 20"
52°C 20"
72°C 5'
repeat 30x
72°C 10'
amplification of pduD
PCR with gradient
PCRmastermix
template
MQ
primer fwd
primer rev
DMSO
sum
D2
8x10µl
8x2µl
8x4.5µl
8x1.5µl
8x1.5µl
8x0.5µl
each20µl
D4
8x10µl
8x4µl
8x2.5µl
8x1.5µl
8x1.5µl
8x0.5µl
each20µl
94°C 2'
94°C 30"
Tx°C 30"
72°C 1'40"
repeat 30x
72°C 5'
charge
D2.1
D2.2
D2.3
D2.4
D2.5
D2.6
D2.7
D2.8
D4.1
D4.2
D4.3
D4.4
D4.5
D4.6
D4.7
D4.8
Tx
45
47.2
52.4
55.2
57.8
60.6
65.8
68
45
47.2
52.4
55.2
57.8
60.6
65.8
68
9-25-2010
9-26-2010
9-27-2010
9-28-2010
9-29-2010
9-30-2010
10-01-2010
PABA
0.5%
1%
2.5%
5%
0%
10-02-2010
10-03-2010
10-04-2010
10-05-2010
10-06-2010
10-07-2010
10-08-2010
10-09-2010
10-10-2010
10-11-2010
10-12-2010
10-13-2010
10-14-2010
10-15-2010
10-16-2010
10-17-2010
10-18-2010
10-19-2010
10-20-2010
10-21-2010
10-22-2010
10-23-2010
10-24-2010
10-25-2010
10-26-2010
10-27-2010
10-28-2010
10-29-2010
10-30-2010
10-31-2010