Team:ULaval/Notebook
Notebook
-gBlock reception and rescue
-Blunt-end cloning – No positives
-Blunt-end cloning – No positives
Figure 1. Typical result of colony PCR for gBlock rescue by blunt-end cloning. Positives should have a band at around 2Kb, higher than what is shown on gel (1Kb NEB DNA ladder). 192 colonies were tested.
-gBlock rescue by PCR.
-gBlock amplification using primers with restriction sequence overhang.
-Sticky end cloning using PCR product – No positives
-gBlock amplification using primers with restriction sequence overhang.
-Sticky end cloning using PCR product – No positives
Figure 2. Initial tests for gBlock rescue using PCR. Initial tests were inconclusive.
Figure 3. Typical result of colony PCR for gBlock rescue by sticky-end cloning. Positives should have a band at around 2Kb, higher than what is shown on gel (1Kb NEB DNA ladder). 288 colonies were tested.
- Design and order of new primers for Gibson assembly.
- gBlock rescue by PCR using newly designed primers.
- Gibson assembly of rescued gBlocks.
- Confirmation and sequencing of obtained clones.
Positives were sequenced to confirm proper insertion and sequence quality. All positive cloned were sequence-confirmed.
- gBlock rescue by PCR using newly designed primers.
- Gibson assembly of rescued gBlocks.
- Confirmation and sequencing of obtained clones.
Figure 4. gBlock rescue using newly designed primers. Wells 1-2: gBlock TH. Wells 3-4: gBlock DDC. Wells 5-6: gBlock DBH. Wells 7-8: gBlock PNMT.
Figure 5. Colony PCR following Gibson assembly and transformation. The presence of a band signifies successful insertion.
Figure 6. Confirmation of positive clones for gBlock insertion. Wells 1-4: gBlock DDC. Wells 5-10: gBlock DBH. Wells 11-16: gBlock PNMT. gBlock TH was confirmed on a separate gel later.
Positives were sequenced to confirm proper insertion and sequence quality. All positive cloned were sequence-confirmed.