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Team:DTU-Denmark/Notebook/24 July 2013
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(→PCR to extract Nir from Pseudosomonas with new USER primers) |
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| - | =208= | + | {{:Team:DTU-Denmark/Templates/StartPage|24 July 2013}} |
| + | Navigate to the [[Team:DTU-Denmark/Notebook/23_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/25_July_2013|Next]] Entry | ||
| + | =Lab 208= | ||
<hr/> | <hr/> | ||
==Main purpose== | ==Main purpose== | ||
<hr/> | <hr/> | ||
| + | * Make USER-reaction to get pZA21 with arabinose inducible promoter. | ||
| + | * Make PCR to get more HAO and AMO with USER-primers. | ||
| + | * Finish the restriction analysis. | ||
==Who was in the lab== | ==Who was in the lab== | ||
<hr/> | <hr/> | ||
| - | Henrike, Julia, Gosia, | + | Henrike, Julia, Gosia, Kristian |
==Procedure== | ==Procedure== | ||
| Line 20: | Line 25: | ||
Reaction is performed in the same way as on [[Team:DTU-Denmark/Notebook/18_July_2013 |18-07-2013]] with increased amount of insert (up to 14 uL due to low DNA concentration). | Reaction is performed in the same way as on [[Team:DTU-Denmark/Notebook/18_July_2013 |18-07-2013]] with increased amount of insert (up to 14 uL due to low DNA concentration). | ||
| + | |||
| + | With the same procedure we also preformed a USER reaction with pZA21::RFP PCR-amplified with no promoter and araBAD from BBa_K808000. | ||
===Restriction analysis=== | ===Restriction analysis=== | ||
| Line 34: | Line 41: | ||
===Primer diluting=== | ===Primer diluting=== | ||
| - | ===PCR to extract Nir from Pseudosomonas with new USER primers=== | + | ===PCR to extract Nir from ''Pseudosomonas'' with new USER primers=== |
Tubes have 4 labels (pZA21 only has 3), in vertical order: | Tubes have 4 labels (pZA21 only has 3), in vertical order: | ||
| Line 40: | Line 47: | ||
*'Nir' or 'pZ', tells which product is being made | *'Nir' or 'pZ', tells which product is being made | ||
*'1' - part 1 of Nir OR '2' - part 2 of Nir OR 'w' - whole Nir (on the pZ tubes 1 and 2 are just duplicates) | *'1' - part 1 of Nir OR '2' - part 2 of Nir OR 'w' - whole Nir (on the pZ tubes 1 and 2 are just duplicates) | ||
| - | *'5' - used 5uL of N. | + | *'5' - used 5uL of ''N. europaea'' culture as template OR '10' - used 10uL of ''N. europaea'' culture as template |
* 'ex' - extraction PCR, using the new non-USER primers that Jakob made OR 'U' - using the new USER primers that Jakob made | * 'ex' - extraction PCR, using the new non-USER primers that Jakob made OR 'U' - using the new USER primers that Jakob made | ||
| Line 49: | Line 56: | ||
part 1 of Nir with USER primers and pZA21 for ligation with Nir - 50C, 5:00 | part 1 of Nir with USER primers and pZA21 for ligation with Nir - 50C, 5:00 | ||
| + | ===PCR with USER-primers on HAO and AMO=== | ||
| + | Standard USER-PCR was made with the HAO and AMO templates purified earlier today. The reactions where done in duplicates and with 2 different concentrations: | ||
| + | *HAO 5 is with 5uL template | ||
| + | *HAO 10 is with 10uL template | ||
| + | *AMO 5 is with 5uL template | ||
| + | *AMO 10 is with 10uL template | ||
| + | |||
| + | All tubes where run with 52C annealing temperature and 3 min extension time. | ||
==Results== | ==Results== | ||
| - | + | <hr/> | |
| - | ==first gel== | + | ===first gel=== |
[[File:2013-07-24 nopro colony extraction.jpg|600px]] | [[File:2013-07-24 nopro colony extraction.jpg|600px]] | ||
| Line 57: | Line 72: | ||
decided to purify RFP in pZA21 without promoter as well as HAO, AMO and cycAX | decided to purify RFP in pZA21 without promoter as well as HAO, AMO and cycAX | ||
| - | ==purification gel== | + | ===purification gel=== |
loaded the complete PCR reaction (~45 uL), cut out the bands of our products and used QIAgen gel extraction kit | loaded the complete PCR reaction (~45 uL), cut out the bands of our products and used QIAgen gel extraction kit | ||
| Line 71: | Line 86: | ||
[[File:2013-07-24 purification.jpg|600px]] | [[File:2013-07-24 purification.jpg|600px]] | ||
| - | ==gel for restriction analysis== | + | ===gel for restriction analysis=== |
[[File:2013-07-24 restriction amo hao cyc.jpg|600px]] | [[File:2013-07-24 restriction amo hao cyc.jpg|600px]] | ||
| Line 78: | Line 93: | ||
==Conclusion== | ==Conclusion== | ||
| + | <hr/> | ||
| + | No of the constructs, Nir, HAO, AMO was shown to have an insert by the restriction analysis. There was only a faint band for the cycAX construct but this have already been verified by colony PCR yesterday and by the orange color of the colonies. We will make a new restriction analysis tomorrow to see if we can locate the insert. | ||
| + | Also we will chech transformant from USER-cloning and possibly make another USER-cloning with the new arabinose inducible vector system. | ||
| - | + | Navigate to the [[Team:DTU-Denmark/Notebook/23_July_2013|Previous]] or the [[Team:DTU-Denmark/Notebook/25_July_2013|Next]] Entry | |
| - | Navigate to the [[Team:DTU-Denmark/Notebook/ | + | |
{{:Team:DTU-Denmark/Templates/EndPage}} | {{:Team:DTU-Denmark/Templates/EndPage}} | ||
Latest revision as of 22:04, 29 September 2013
24 July 2013
Contents |
Lab 208
Main purpose
- Make USER-reaction to get pZA21 with arabinose inducible promoter.
- Make PCR to get more HAO and AMO with USER-primers.
- Finish the restriction analysis.
Who was in the lab
Henrike, Julia, Gosia, Kristian
Procedure
USER reaction and transformation
We perform USER reaction, samples are as follows:
- plasmid pZA21 and AMO
- plasmid pZA21 and HAO
- negative control with water instead of insert
Reaction is performed in the same way as on 18-07-2013 with increased amount of insert (up to 14 uL due to low DNA concentration).
With the same procedure we also preformed a USER reaction with pZA21::RFP PCR-amplified with no promoter and araBAD from BBa_K808000.
Restriction analysis
From last week transformants with AMO and HAO in pZA21 we performed plasmid isolation.
We perform restriction analysis with EcoRI. Expected fragments are as follows:
- For pZA21 with AMO:
3309 pz, 2283 pz
- For pZA21 with HAO:
2233 pz, 2124 pz, 930 pz
Primer diluting
PCR to extract Nir from Pseudosomonas with new USER primers
Tubes have 4 labels (pZA21 only has 3), in vertical order:
- 'Nir' or 'pZ', tells which product is being made
- '1' - part 1 of Nir OR '2' - part 2 of Nir OR 'w' - whole Nir (on the pZ tubes 1 and 2 are just duplicates)
- '5' - used 5uL of N. europaea culture as template OR '10' - used 10uL of N. europaea culture as template
- 'ex' - extraction PCR, using the new non-USER primers that Jakob made OR 'U' - using the new USER primers that Jakob made
two programs:
all extraction PCRs and part 2 of Nir with USER primers - 54C, 5:00
part 1 of Nir with USER primers and pZA21 for ligation with Nir - 50C, 5:00
PCR with USER-primers on HAO and AMO
Standard USER-PCR was made with the HAO and AMO templates purified earlier today. The reactions where done in duplicates and with 2 different concentrations:
- HAO 5 is with 5uL template
- HAO 10 is with 10uL template
- AMO 5 is with 5uL template
- AMO 10 is with 10uL template
All tubes where run with 52C annealing temperature and 3 min extension time.
Results
first gel
decided to purify RFP in pZA21 without promoter as well as HAO, AMO and cycAX
purification gel
loaded the complete PCR reaction (~45 uL), cut out the bands of our products and used QIAgen gel extraction kit
- 1: 1 kb ladder
- 2: HAO
- 3: AMO
- 4: cycAX
- 5: RPF in pZA21 without promoter
- 6: RPF in pZA21 without promoter
- 7: RPF in pZA21 without promoter
gel for restriction analysis
Conclusion: We only get one band for each plasmid, so the inserts are not present.
Conclusion
No of the constructs, Nir, HAO, AMO was shown to have an insert by the restriction analysis. There was only a faint band for the cycAX construct but this have already been verified by colony PCR yesterday and by the orange color of the colonies. We will make a new restriction analysis tomorrow to see if we can locate the insert. Also we will chech transformant from USER-cloning and possibly make another USER-cloning with the new arabinose inducible vector system.
Navigate to the Previous or the Next Entry
