Team:Toronto/Notebook-w11-wed
console.js:26
Wednesday, July 27
Wednesday, 7/27
Members Present: Hamed, Kat, Tam, Cathy, Alex, Bohdan (for morning)
LAB:
Morning:
●
RE Single Digest was performed on the following miniprepped samples:
■
RFP + A1
■
RFP + A2
■
RFP + A3
■
RFP + B1
■
RFP + B2
■
RFP + B3
■
RFP - A1
■
RFP - A2
■
RFP - B
■
Kit B
■
CjBlue
■
Negative/Nuclease Free Water
○
Each sample contained except negative contained:
■
2ul of NEB Buffer 3.1
■
1ul of NEB Pst1
■
15.9ul of KitB, 14.9ul of CjBlue, 16ul for the rest except negative
■
1.1ul of NF Water for KitB, 2.1ul of NF Water for CjBlue, 1ul of NF Water for the rest
■
Final volume of 20ul
○
For negative/nuclease free water, it contained:
■
2ul of NEB Buffer 3.1
■
1ul of NEB Pst1
■
17ul of NF Water
Afternoon:
●
Ran gel on the RE single digested samples (All gels used 8ul of sample with 1.8ul of loading dye and 6ul of NEB 2-log DNA ladder which ran at 100V for 60 min):
○
Gel 1:
○
Gel 2:
Lane 1: RFP-A1, single band between 2kb and 1.5kb
Lane 2: RFP-A2, single band below 1.5kb
Lane 3: RFP-B, single band below 1.5kb
Lane 4: NEB 2-log DNA Ladder
Lane 5: Kit+B, two bands (below 4kb and between 3kb & 2kb)
Lane 6: CjBlue, single band at ~3kb
Lane 7: Negative control/Nuclease Free Water, no bands
●
Realized that the reason why our ligations weren't working may have been buffer incompatability and a lack of PCR purification - notably, our PCR Phusion was from Thermo, and our subsequent restriction enzymes (and everything else) is from NEB. It could be that a high salt concentration from the PCR (from Mg+ ions - required as a cofactor for polymerase) actually inhibit the RE functioning. Either way, we will proceed with Phusion from NEB, and add an additional PCR purification step between PCR and RE digest.
●
Also recieved some insert and backbone from Kayla, to troubleshoot our plasmid.
Administrative:
●
Meeting with OG was organized with Anthony, Alex and Ben for Friday, July 29th at 1400
