"
Team:Groningen/1 June 2010
From 2010.igem.org
(Difference between revisions)
| Line 3: | Line 3: | ||
We've started working on the wiki, been preparing plasmids and are settling in our ''office space''. Note to self: should have brought coffee machine | We've started working on the wiki, been preparing plasmids and are settling in our ''office space''. Note to self: should have brought coffee machine | ||
| - | < | + | <pre>Week 22 |
| - | + | ||
| - | The biobrick prefix and suffix are introduced into the pNZ8901 expression vector using PCR and primers with 5’ overhangs. For optimization taq polymerase (fermentas) is used first | + | The biobrick prefix and suffix are introduced into the pNZ8901 expression vector using PCR and primers with 5’ overhangs. |
| - | + | ||
| - | + | For optimization taq polymerase (fermentas) is used first | |
| + | Component µl Final concentration | ||
Primer pNZ89bbs-for1 5 300nM | Primer pNZ89bbs-for1 5 300nM | ||
Primer pNZ89bbs-rev1 5 300nM | Primer pNZ89bbs-rev1 5 300nM | ||
| Line 16: | Line 16: | ||
Template 0.5 ~14ng | Template 0.5 ~14ng | ||
Taq 0.5 2.5u | Taq 0.5 2.5u | ||
| - | MQ 29 | + | MQ 29 |
- 94°C, 3’ | - 94°C, 3’ | ||
| Line 24: | Line 24: | ||
- 72°C, 10’ | - 72°C, 10’ | ||
| - | + | No product. Likely the elongation step was too short for taq. | |
| - | + | ||
| + | Repeat of previous PCR using the following cycling conditions | ||
| + | |||
| + | - 94°C, 3’ | ||
| + | - 94°C, 30’’ | ||
| + | 30X - 50°C, 30’’ | ||
| + | - 72°C, 3’ | ||
| + | - 72°C, 10’ | ||
| + | |||
| + | Gel 02-06-10 | ||
| + | Product is ~4kb instead of 3,2. This has been observed before and plasmid is ok to use. | ||
| + | Repeated PCR with pfu polymerase (fermentas) to prevent point mutations in plasmid</pre> | ||
Revision as of 21:40, 7 October 2010
Hydrophobofilm
pushing coatings into a greener future
We've started working on the wiki, been preparing plasmids and are settling in our office space. Note to self: should have brought coffee machine
Week 22 The biobrick prefix and suffix are introduced into the pNZ8901 expression vector using PCR and primers with 5’ overhangs. For optimization taq polymerase (fermentas) is used first Component µl Final concentration Primer pNZ89bbs-for1 5 300nM Primer pNZ89bbs-rev1 5 300nM 10x Taq buffer(-MgCl2) 5 1x dNTP’s 2 200µM MgCl2 3 1.5mM Template 0.5 ~14ng Taq 0.5 2.5u MQ 29 - 94°C, 3’ - 94°C, 30’’ 30X - 50°C, 30’’ - 72°C, 1’30’’ - 72°C, 10’ No product. Likely the elongation step was too short for taq. Repeat of previous PCR using the following cycling conditions - 94°C, 3’ - 94°C, 30’’ 30X - 50°C, 30’’ - 72°C, 3’ - 72°C, 10’ Gel 02-06-10 Product is ~4kb instead of 3,2. This has been observed before and plasmid is ok to use. Repeated PCR with pfu polymerase (fermentas) to prevent point mutations in plasmid
Week 23
