Team:UCLA/Notebook/Interlab Study/20 August 2015
= Results of Transformation & Colony PCR screening +
Results of Transformation
Redo of the transformation using Tuesday's protocol seemed to work, presence of large growth of individual colonies formed on the plates. 1:1 dilutions showed expansive lawn growth of colonies, whereas the 1:10 dilution showed growth of fewer, but larger colonies. 1:!0 dilutions showed little to no colony formation, suggesting higher transformation effect in the 1:10 range. Images of plates will be posted via Benchling.
For each of the devices, 4 colonies were picked from the three ligation 1:1 plates. Each colony was circled and labeled, as showing the plate group below.
To screen for proper insert size and presence, colony PCR was conducted using RedTaq master mix and VF2/VR primers. The following protocol was used.
First, each colony was picked and suspended in 50 uL of sterile LB using a sterile tip. The suspension was left at RT during the day to later be used to inoculate overnight cultures of sequencing and stocking.
| Component | Amount (12.50 uL RXN) |
|---|---|
| RedTaq PCR Ready Master Mix | 6.25 uL |
| Colony DNA Template | 1uL cell suspension |
| VF2 FWD Primer (pSB1C3) | 0.625 uL |
| VR REV Primer (pSB1C3) | 0.625 uL |
| Nuclease Free ddH2O | 4 uL (up to 12.50 uL) |
A negative control was generated using 1 uL of sterile LB instead of suspended colony. Each colony and control was prepared in a separate PCR tube, mixed, and centrifuged. Samples were loaded into a thermocycler with the following settings in a 13 uL reaction condition:
| STEP | TEMP (degrees Celsius) | TIME |
|---|---|---|
| Denaturation & Cell Lysis | 95 | 3 minutes |
| Denaturing (30 cycles) | 95 | 25 seconds |
| Annealing (30 cycles) | 56 | 30 seconds |
| Extension (30 cycles) | 72 | 2 minutes |
| Final Extension | 72 | 5 minutes |
| Hold | 4 | Infinitely |
Samples were then removed from thermocycler, chilled on ice, and loaded directly into a 1% wt/vol agarose gel with 1X SYBRsafe (10 uL of 10,000x stock in 100mL of 1g agarose gel in 1x TAE). Gel was ran for 40 minutes at 150 V, with dilution of NEB 1kb Ruler (2 uL ruler, 8 uL nuclease free water, 2 uL 6x NEB loading dye). Gel was then removed and imaged in Bio-Rad Image Lab for screening.
Results of Colony PCR
Colony PCR results indicate a positive screen for all 4 colonies in each device transformation. Some nonspecific binding does occur, but opportunistic binding and amplification of a 1 kb average insert suggests presence of the insert in between the VF2/VR binding regions. This correlated with an insert approximately 950 bp in length with added overhangs, which is what we expect for an insert of the device length.
However, the negative control was disconcerting, with a 1.5 kbp band generating. No band was expected in the negative control, suggesting template contamination of the LB, water, master mix, primers, or equipment. Care must be taken to decontaminate and discard all contaminants from now on. However, specific binding of the insert at the correct size in the clones indicates proper presence of device in each ligation product. Forever, GFP screening using the Invitrogen gel box aided in screening colonies, electing those that fluoresced under standard conditions (no the case for Device 3, but for Devices 1 and 2).
Next steps
Three colonies were selected from each device (1-1, 1-2, 1-4, 2-1, 2-2, 2-3, and 3-1,3-2,3-3), excluding one colony based on purity of the band and potential for differential insert via non-specific binding.
Each corresponding 49 ul suspension was use to inoculate 10 mL of LB with chloramphenicol (34 ug/uL), and placed O/N in a 200 RPM shaking incubator at 37 degrees. A negative control using the sterile LB of interest in the colony PCR was placed in the same media and incubator O/N.
OD will be checked for all cultures tomorrow with 1mL of culture. Additionally, 500 uL will be used to glycerol stock each clone. 3 mL will be used to miniprep the device plasmids and small aliquots will be taken for sequencing. The remains will be diluted and used in 96 well format for fluorescence practice assaying using the BioTex plate reader and software.
Refer to tomorrow's wiki for more information.