Team:Groningen/Notebook/21 September 2009

GVP Cluster
Planning


 * → work out the wiki page for GVP
 * made a layout
 * still have to read all articles
 * modeling will stay as it is, has been done by modeling people
 * → ✅ make a doodle for presentation planning (1-19 oct.)
 * → ✅ media attention
 * mail to UK, Ing., St. Gen.
 * facebook account with link to twitter
 * ethics survey link on facebook and twitter
 * → ✅ place an ethics survey link on twitter


 * → ✅ clone pArsR-GVP into pSB2K3
 * still need to make glycerol stock
 * → clone repeat out of GVP cluster
 * → make glycerol stocks of constructs
 * → enter info on part registry

Restriction for Assembly

The plasmid of pMA-gvpL insert was cut with MvaI/XhoI to cut out the entire part of wanted fragment.

Purification




 * → In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use.


 * → The concentration of GVP fragment with X/P sticky ends was 81.9 ng/μL, and was stored in fridge for restriction by MvaI/XhoI on Tuesday.

Ligation

A total amount of vector of 100ng was used in a 1:3 ratio with insert.
 * 3 uL Ligase buffer
 * 1 uL T4 Ligase
 * 3 uL GVP fragment 3300bp
 * 3 uL GVP fragment 2200bp
 * 4 uL plasmid J61035 XbaI/PstI
 * 8 uL GVP-insert (restricted with MvaI/XhoI)

Incubate:
 * 25°C 50min.
 * kept on ice for 10min.

Tranformation
 * add 10uL of the ligation product to 50uL competent E.coli TOP10 cells.

Incubate:
 * 30 min @ ice
 * 90 sec 42°C
 * 2 min @ ice
 * add 800uL LB-medium
 * incubate for 1 h at 37°C
 * plate on LB-amp100 plates


 * → Negative control was MQ and positive was J61002-J23101.

Cultures

From the plates of last friday (growth from thursday to friday) two colonies of pMA-gvpL-insert, pMA-GlpF and pNL29, and one colony of pArsR-GVP in pSB2K3 were used to inoculate 6mL LB-amp100 and LB-kan50-IPTG medium for growth overnight.


 * → The tubes were stored in a 37°C shaking waterbath.

Transporters
HmtA

Nieuwe poging om HmtA te krijgen. Via verschillende routes, PCR1 met F1 mut1rc en EcoRI cut pBAD met 742 f2 en PCR1 & 2.