Team:Stockholm/13 August 2010
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NinaSchiller (Talk | contribs) (New page: {{Stockholm/Top2}} ==Nina== ---- ==Gel verification of protein A (ZZ domain) and IgG protease== I ran a new agarose gel 1 % on my protein A and IgG protease samples. However I didn't lo...) |
NinaSchiller (Talk | contribs) (→Gel verification of protein A (ZZ domain) and IgG protease) |
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[[Image:Gel1.jpg]] | [[Image:Gel1.jpg]] | ||
[[Image:Prot.jpg|250px]] | [[Image:Prot.jpg|250px]] | ||
+ | |||
+ | ---- | ||
+ | ==Colony-PCR of site-directed mutagenesis Tyrosinase== | ||
+ | |||
+ | Colony nr: 2, 4, 6 & 8. | ||
+ | |||
+ | PCR Mix: | ||
+ | |||
+ | Total volume 100 ul, aliquate 25 ul in the four pcr tubes. | ||
+ | |||
+ | *F primer 50 uM 1 ul | ||
+ | *R primer 50 uM 1 ul | ||
+ | *dNTP 10 uM 1 ul | ||
+ | *Buffer phusion 5X 20 ul | ||
+ | *Phusion polymerase 1 ul | ||
+ | *H2O 75 ul | ||
+ | |||
+ | PCR prgm: | ||
+ | |||
+ | 98 °C 2 min | ||
+ | |||
+ | 22 cycles of: | ||
+ | |||
+ | *98 °C 30 sec | ||
+ | *50 °C 30 sec | ||
+ | *72 °C 2 min | ||
+ | |||
+ | 72 °C 1 min | ||
+ | |||
+ | 4 °C ∞ | ||
+ | |||
+ | I ran 5 ul of the PCR products with 1 ul loading dye 6X on an agarose gel 1 % 80 V. | ||
+ | |||
+ | Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas | ||
+ | |||
+ | Arrangement on gels: | ||
+ | |||
+ | [[Image:Tyr-site.jpg]] | ||
+ | |||
+ | [[Image:Tyr.jpg|250px]] | ||
+ | |||
+ | ---- | ||
+ | ==Overnight culture of protein A (ZZ domain) and Tyrosinase== | ||
+ | |||
+ | *I inoculated protein A ZZ domain inserted in the bank vector C (with chloramphenicol) colony # 5 in 12 ml LB and 24 ul chloramphenicol 50 mg/ml. This was incubated O/N in 37 °C with shake. | ||
+ | |||
+ | *I inoculated tyrosinase in its original vector colonies # 2, 4, 6 & 8 in 12 ml LB and 24 ul ampicillin 50 mg/ml. This was incubated O/N in 37 °C with shake. | ||
+ | ---- | ||
+ | ==Sequencing of protein A (ZZ domain)== | ||
+ | |||
+ | I prepared a tube of protein A ZZ domain for sequencing: | ||
+ | |||
+ | 15 ul plasmid from a mini-prep and 1.5 ul (10uM) primer. Preferably the forward primer (VF) of the vectors verification primers. | ||
+ | |||
+ | * ASB0045 303 |
Revision as of 21:40, 13 August 2010
Contents |
Nina
Gel verification of protein A (ZZ domain) and IgG protease
I ran a new agarose gel 1 % on my protein A and IgG protease samples. However I didn't load the positive protein A control sample since I realized that it would not fullfill its purpose considering the vector verification primer I used does not bind to the vector protein A sits in, which is the original vector I obtained it in.
Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas
Arrangement on gels:
Colony-PCR of site-directed mutagenesis Tyrosinase
Colony nr: 2, 4, 6 & 8.
PCR Mix:
Total volume 100 ul, aliquate 25 ul in the four pcr tubes.
- F primer 50 uM 1 ul
- R primer 50 uM 1 ul
- dNTP 10 uM 1 ul
- Buffer phusion 5X 20 ul
- Phusion polymerase 1 ul
- H2O 75 ul
PCR prgm:
98 °C 2 min
22 cycles of:
- 98 °C 30 sec
- 50 °C 30 sec
- 72 °C 2 min
72 °C 1 min
4 °C ∞
I ran 5 ul of the PCR products with 1 ul loading dye 6X on an agarose gel 1 % 80 V.
Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas
Arrangement on gels:
Overnight culture of protein A (ZZ domain) and Tyrosinase
- I inoculated protein A ZZ domain inserted in the bank vector C (with chloramphenicol) colony # 5 in 12 ml LB and 24 ul chloramphenicol 50 mg/ml. This was incubated O/N in 37 °C with shake.
- I inoculated tyrosinase in its original vector colonies # 2, 4, 6 & 8 in 12 ml LB and 24 ul ampicillin 50 mg/ml. This was incubated O/N in 37 °C with shake.
Sequencing of protein A (ZZ domain)
I prepared a tube of protein A ZZ domain for sequencing:
15 ul plasmid from a mini-prep and 1.5 ul (10uM) primer. Preferably the forward primer (VF) of the vectors verification primers.
- ASB0045 303