Team:NYU Gallatin/Notebook/Notes

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Lab Notes

Author Team Date Note Edit link
Steven Cloning Aug/15/2012

The next day we had lots of colonies (I don't have the pictures of the plates, but maybe someone else does). Many of these were red, indicating they were transformed due to residual RFP plasmid (as had happened previously). We circled several white colonies as prospective positive transformants. For each construct, we chose 5 clones and inoculated 4 ml of LB-chlor to grow these overnight at 37 deg C.
Steven Cloning Aug/14/2012

The second gibson assemblies that we did worked! We followed Ellen's advice to increase the G-bit DNA concentration. This means our final assembly reactions looked like this:

2.5 ul pSB1C3 (biobrick backbone plasmid DNA, linearized)
7.5 ul G-bit DNA*
10 ul master mix
20 ul total

* we divided the volume left by the number of G-bit parts. For NAG1, there were 3 parts, so each took up 2.5 ul. For UAP 1, there were 4 parts, so each took up 1.8 ul. For AGM1, there were 5 parts, so each took up 1.5 ul.

We incubated these for 1 hr at 50 deg C and then immediately transformed the commercial competent cells.
Steven Cloning Aug/12/2012

Gibson, Transformation:
So instead of 50 fentomoles of each, try 100 but 50 of the plasmid as before. I think we have room in the 10 microliters for that. And maybe close the lid of the PCR machine?

Gibson assembly protocol

-reaction is in 0.2mL PCR tube using program �Gibson� under user �ellen�
50C for 1h, hold at 4C
-total reaction volume is 20 microliters
-all G-Blocks were resuspended at a concentration of 50 fentomoles per microliter
-TOTAL amount of DNA in tube (sum of all parts) should be between 200-1000 fentomoles. NOTE: last time we were at the low end of this!

Recommended procedure:
For �U�
1.85 µL U-1
1.85 µL U-2
1.85 µL U-3
1.85 µL U-4
2.6 µL pSB1C3
10.0 µL Gibson Master Mix
20.0 µL

For �N�
2.5 µL N-1
2.5 µL N-2
2.5 µL N-3
2.5 µL pSB1C3
10.0 µL Gibson Master Mix
20.0 µL
Alex Cloning Aug/10/2012

Results of Gibson Assembly:
4 big plates of the 3 chitin path genes A, U, and N, a + control
2 small plaets: neg control, baseline control without antibio

Results:

Lawn on antibiotic free control

Some contamination on A & U plates

No growth on N, + Control plates

[PASTE ALL PHOTOS FROM ALEX EMAIL AFTER RESIZING]

Pink colonies may be traces of RFP plasmids remaining from when biobrick plasmid part was PCR�ed.

Reasons for failure could be:

1) use the heated lid on the PCR machine - I did not shut it because I thought it might jack up the temp.
2) the manual says to use a 2-3x overage of inserts to backbone. The tech service guy said use equimolar so we did. maybe we need to rethink that.
3) overall total conc of DNA in the rxn mix could be upped.
Alex Cloning Aug/08/2012

Notes for NAG1 Gibson

N-1 87163240
163696916 Date Ordered: 3rd Aug, 2012
200 ng = 1012.2 fmol

N-2 87163241
163696911 Date Ordered: 3rd Aug, 2012
200 ng - 1005.9 fmol

N-3 87163242
163696921 Date ordered: 3rd Aug, 2012
200 ng = 978.5 fmol

Gibson Assembly Master Mix
#E26115 Exp: 6/13
Lot: 0031206

PSBIC3
Biobrick Plasmid 2071 bases
650 * 2071 =
Mol. Weight: 1.346 x 106 fg/fmol

N1
10012.2 fmol / 20 μL
= 50.61 fmol/ μL
N2
1005.9 fmol / 20 μL
= 50.29 fmol/ μL
N3
978.5 fmol / 19 μL
= 51.50 fmol/ μL

4.3 μL water
+ 2.7 μL PSB1C3
+ 1.0 μL N1 (Concentration - 50.61 fmol/μL)
+ 1.0 μL N2 (Concentration - 50.29 fmol/μL)
+ 1.0 μL N3 (Concentration - 51.50 fmol/μL)
+ 10 μL Mastermix
= 20 μL
Steven Cloning Aug/06/2012

G-blocks arrived
Sara Growing Jun/09/2012
  1. Almost no growth, very thin film.
  2. Cellulose very thin, seems to have grown on bottom (not top)
  3. Thin growth, not measurable from sides, but def more significant than just scoby.
  4. Super thick from bubbles, accidentally popped it. A little thicker than #9.
  5. Infested w/ flies. Nice growth, not in a sheet, disposing.
  6. Pretty thick, made a little pillow from the air bubbles.
  7. Thin growth along top.
  8. Thin growth along top.
  9. Nice sheet, 2nd to 10+13.
  10. Very thick cellulose, abt 1-2mm growth.
  11. Thickest non-BB. Weird flakes (brown) everywhere.
  12. Thin growth, a little thicker than the other thins.
  13. Almost entirely cellulose, very little liquid left.

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