Aim of the construct

• We'll try out 35s->GAL4->HAP1GR->H2BmRFP.
• Since H2BmRFP has not been tried in Mp, we'll also try 35s->H2BmRFP. A positive control for lab technique will be 35s->Venus N7
• We'll also use FF's original plasmid to try 35s-Hap1GR-mRFP, as a control for the Hap1GR. I expect low transformation rate as in the original plasmid, we're using 35s as promoter for HygR selection marker.
• Not sure whether 35s->Gal4 has been tried (check with BP), so 35s-Gal4-venus n7 is doable with BP's construct as a control
• As another control, we'll try 35s-Gal4-H2mRFP

End goal plasmid

• 

PCR

Attempt 1:

• Start date/time: 24/07, 11:08
• End date/time:24/07, 1550
• UIDs of primers/plasmids used:
  • A1, P33, P34
  • A1, P35, P36
  • A2, P37, P38
  • A2, P39, P40
• PCR Spreadsheet (on google drive)
• Gel 27/07/2014

Gibson

Attempt 1:

• Start date/time: 25/07
• Comments: seemed to be fine.

E-Coli transformation

Attempt 1:

• Start date/time: 25/07
• Comments:

Negative control negative, positive control positive.

Selection

Attempt 1:

• Start date/time: 28/07
• Comments:

Selected initially just 3 colonies. Grew them in LB and miniprepped on 27/07. Performed digest with SalI to check for template carryover and see that assembly was done. DEXX3, pb>RVe each have a unique site. Following digest with Sal1, we should have a 12kb fragment and a 2kb fragment. After digest, 2kb fragment was visible, but 12kb fragment was not. Very mysterious.
Some error in the digestion protocol is likely to have occurred. To insert a promoter, a digest with Kpn1 was performed. Gel was illegible, although a faint band at 13kb may have been visible. Attempt 2:

• Start date/time: 30/07
• Comments:

Grew up in LB 8 more colonies. Attempted colony PCR using inoculation loop in dry PCR tube - will wait to see results from GEL. Expect an 8kb fragment: unlikely that this will show results. Will perform a SalI digest with correct protocol on 31/07 following miniprep from colonies.

Agrobacteria transformation

Attempt 1:

• Start date/time:
• End date/time:
• Induce comments:
• Growth comments:
• Electroporation/selection comments:

Spore preparation

Attempt 1:

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Spore transformation

Attempt 1:

• Start date/time:
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Evaluation

• Start date/time:
• End date/time:
• Comments: