Team:Toronto/Notebook-w11-thu
console.js:26
Thursday, July 28
Thursday, 7/28
Members Present: Hamed, Kat, Alex, Tam, Karim, Marc, Bohdan
LAB:
Morning:
●
Gradient PCR amplification of the pSB1C3 backbone from RFP using NEB's Phusion polymerase. Gradient was run high-low with corresponding samples B (A-F)
●
Gradient PCR amplification of the Short TetO LacZ insert using NEB's phusion Polymerase. Gradient was run high-low with corresponding samples Z (A-F)
●
Prepped our bio-basic PCR purification kit (using anyhdrous alcohol for the wash buffer)
Afternoon:
●
Ran two gels on our gradient PCR of the pSB1C3 and on the LacZ:
Gel on the gradient PCR of pSB1C3 backbone using NEB's Phusion polymerase.
Lane 1: GolS/P118A?
Lane 2: BA
Lane 3: BB
Lane 4: 2-log ladder
Lane 5: BC
Lane 6: BD
Lane 7: BE
Lane 8: BF Note that because the strongest bands were obtained with BA and BB (highest temperatures), only those samples were kept and the respt were discarded
●
PCR purification and nanodrop of the (successfully) PCR'd constructs:
A | B | C | D | |
1 | SAMPLE | 260/280 | 260/230 | CONC (ng/uL) |
2 | ZE | 1.88 | 1.86 | 56.1 |
3 | ZF | 1.88 | 1.81 | 69.3 |
4 | BA | 1.86 | 2.05 | 85.6 |
5 | BB | 1.89 | 1.37 | 100.2 |
6 | d1 | 1.68 | 1.13 | 10.7 |
7 | d2 | 1.61 | 0.87 | 23.4 |
8 | GolS | 1.83 | 2.03 | 55.1 |
9 | GolSP118A | 1.78 | 1.97 | 56.5 |
10 | 5 | 1.65 | 0.86 | 33.5 |
11 | 3 | 1.69 | 0.9 | 20.2 |
Table2
Nanodrop results selectively from the successfully PCR's samples. For the LacZ-alpha gradient PCR, less concatermerization was seen at lower temperatures, corresponding to rows E and F. For the bacbone, successful PCRs were at the higher temperature range, corresponding to A and to B. d1 and d2 are the gel extracted backbone. GolS and GolSP118A are both PCR products from the previous day, done using Q5 polymerse from NEB. Sample 5 is a previous amplication using Short GolS P118A. Sample 3, similarly is amplified pgolB. Both 5 and 3 were done using Thermo's Phusion.
●
Inputted the concentrations into out ligation calculator and proceeded with RE digest and heat kill. For time-efficiency, dpnI was included in the backbone digest. These are the samples we digested:
○
Insert (short TetO lacZ-alpha)
○
Backbone digested
○
Backbone undigested (identical to digested, just that the enzymes are replaced with H20)
○
Backone dpnI (this is gel extracted backbone, that has been dpnI digested O/N)
●
rSAP of the backbone digest for 30 mins followed by heat kill
●
PCR purification following the digest.
●
Nanodrop of the PCR purified and digested constructs (as well as the constructs provided by Kayla):
A | B | C | D | |
1 | SAMPLE | 260/280 | 260/230 | CONC (ng/uL) |
2 | BB digested | 1.66 | 1.05 | 40.6 |
3 | BB undigested | 1.63 | 0.94 | 44.2 |
4 | BB dpnI | 2.29 | 10.97 | 8.5 |
5 | insert (lacZ) | 1.7 | 2.45 | 20.2 |
6 | HISB (Kayla's insert) | 3.12 | 0.75 | 24.1 |
7 | pCOLA (Kayla's backbone) | 1.88 | 0.24 | 3.5 |
8 | Kayla's plasmid | 2.12 | 1.79 | 57 |
Table3
●
Inputted the concentrations into our ligation calculator, and proceeded to Ligate the following:
○
Insert-backbone digested
○
Insert-backbone undigested
○
insert-backbone dpnI
○
HisB - pCOLA plasmid
●
Ligate O/N for 16hrs
Today's ligation calculations for reference:
Administrative:
●
WB407 booked for Friday, July 29, 2016 for use of a P&P meeting during 1800-2000
A | B | C | D | E | F | |
1 | Name | SKU | Unit Price | Quantity | Total (incl. tax) | Status |
2 | Phusion High-Fidelity DNA Polymerase | M0530S | 117.38 | 100 units | 132.64 | available =>in Yuki door |
Table1
TO DO:
For the next day:
LAB TEAM:
●
Ask Kayla if her pCOLA plasmid has a Kan (Kanamycin) resistance marker, and if yes, then if she could provide us with two plates?
●
Transformation with:
○
Ligated constructs:
■
digested
■
undigested
■
dpnI
■
pCOLA
○
Kayla's pCOLA plasmid
●
Remember to transform with 5ul of product. Use CCD cells in fridge.
