Team:Wageningen UR/Journal/week19
From 2012.igem.org
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When he finally returned on tuesday, there was no difference. | When he finally returned on tuesday, there was no difference. | ||
- | + | '''Hepatitis B ouside modification''' | |
* 7 September (Kees) | * 7 September (Kees) |
Revision as of 08:57, 13 September 2012
Mark's hair
Mark will get married soon, so he had to spend the entire monday having his hair done. When he finally returned on tuesday, there was no difference.
Hepatitis B ouside modification
- 7 September (Kees)
• 5’phosphorylated primers
By amplifying the whole plasmid, including the whole desired insert, only one pcr step is needed which can be used right away to transform bacteria.
The sequence: The Primers:
Legend: Blue: HepBcAg gene Arrows: primers Black: Backbone Red Star: 5’phosphorylation on primers and DNA
The primers used for whole plasmid pcr arrived and were diluted to 100µM.
The PCR mixture:
component Volume (µL)
Primers (FW and rev) 2
Enzyme (Phusion Thermo) 0.5
DMSO 100% 1
Buffer (HF Thermo) 10
Template (20ng/µL) 1
MQ water 34
dNTPs 1
PCR protocol: 1: 98˚C; 2min 2: 98˚C; 30s 3: 63˚C; 30s 4: 72˚C; 30s 15× from step 2 5: 4˚C; ∞ After the PCR, 10µL was used to digest with DpnI, 10µL was mixed with T4 ligase and the rest left untreated. DpnI was heat inactivated after which all three different samples (Ligase, DpnI, normal) were used to transform DH5α.
- 8 September (Kees)
• Colonies
All plates contained colonies, however there was only one on the DpnI-digested plate, which would suggested incomplete digestion rather than a wanted colony. Therefore, more of the sample was plated in order to obtain more colonies.
- 9 September (Kees)
• Checked the plates
There were no new colonies at all on the two new plates.