Team:GeorgiaTech/WeekTwo

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8/9/2010
Ligation
Purpose: Insert mRFP into pCold vector

In a microcentrifuge tube add:
1. vector DNA (pCold)
2. insert DNA (mRFP)
3. Ligase 10X buffer
4. T4 DNA Ligase
5. Nuclease-free water to a final volume of 10սL.
6. Incubate reaction at 4C overnight.

*(pCold: 5700 bp; mRFP: 700 bp)

Vector:insert	1:1	1:2	1:4	1:8
pCold (սL)	2	2	2	4
mRFP (սL)	3	4.7	3	3
10X buffer (սL)	1	1	1	1
T4 Ligase (սL)	0.5	0.5	0.5	0.5
dH2O (սL)	3.5	1.8	3.5	1.5
total (սL)	10	10	10	10

Making LB media
To make 1L LB (Lysogeny broth) medium:
5g Yeast abstract
10g Tryptone
5g NaCl

To make solid plates, follow the above recipe and add 16g agar per 1L.

Prepared 2L of LB media, 1L liquid broth and 1L to make plates (don’t fill 1L bottles full - make 4 bottles 500 mL each).
Autoclave, leaving lids slightly loosened.
Let cool in water bath at 55C.
Add antibiotic to LB media used for plates.
For the agar plates, pipetted 25mL into each plate

Incubating reactions
The bacteria were left at 4C overnight.

8/10/2010

Heat shock transformation of the plasmids into our bacteria
10 սL Nova Blue cells + 5 սL of ligation rxn

1. Left cells and ligation reaction products on ice.
2. Added plasmid (5սL) (ligation product) to cells. Mix gently by swirling pipet tip in mixutre (DO NOT ASPIRATE).
3. Left cells on ice for 10-30 min.
4. Applied heat shock of 45 seconds in 42C bath.
5. Put tubes on ice for 2 min.
6. Added 250 սL of LB (room temp.)
7. Incubated 1 hour at 75C (9/1 Edit: Seems improbably high- probably 37 C)
8. Plated 100սL, then remaining amount on separate LB/CARB plates.
9. Incubated overnight at 37C.

8/11/2010

Inoculation

1. Add 5mL LB and 5սL CARB into test tubes (16 total)
2. Pick 2 well-defined colonies from each plate (labelled sequentially).
3. With an inoculation loop, get 1 colony from transformation plate and place into test tube.
4. Repeat for each colony (16 total).
5. Inoculate overnight at 37C.

8/12/2010

Miniprep

1. Remove inoculation tubes from inoculation (37C shaker).
2. Obtain P1 buffer from 4C refrigerator.
3. Take centrifuge tubes and add 1.5mL on inoculated cells.
4. Centrifuge at 3000 rpm (low) for 1-2 min.
5. Spin until white pellet of cells forms at the bottom and liquid is more clear.
6. Take off supernatant and discard.
7. Repeat steps 4-6.
8. Resuspend pelleted bacterial cells in 250սL P1 buffer.
9. Add 250սL P2 buffer and invert 4-6 times (DO NOT VORTEX - doing so will shear DNA!)
10. Add 350սL buffer N3 and immediately invert 4-6 times.
11. Centrifuge for 10 min. at 13,000 rpm.
12. Take supernatant and add to spin columns.
13. Spin 30-60 sec. and discard flow through.
14. Wash column with 750սL buffer PE and centrifuge 1 min.
15. Discard flow through and centrifuge and additional minute.
16. Please column into a clean 1.5mL microcentrifuge tube.
17. Elute DNA by adding 30սL dH2O.
18. Let stand for 1 min., then centrifuge for 1 min.

Double digest

Into an Eppendorf tube add:
1. ~ 25սL DNA
2. 10սL autoclaved H2O.
3. 4սL 10X buffer.
4. 0.5սL Nde1 enzyme.
5. 0.5սL Xho1 enzyme.

8/13/2010

Making gel for PCR
1. Add 0.5g agar to 50mL autoclaved water.
2. Add 5mL 1X TBE
3. Allow to cool.
4. Add 45սL EtBr
5. Pour gel and allow to harden.

Running PCR
1. Fill gel to top with 1X TBE.
2. Take 3սL loading dye and make a spot on parafilm.
3. Add 10սL DNA to dye on parafilm. Pipette up and down to mix (NO BUBBLES).
4. Load 5սL of ladder at each end.
5. Add DNA + dye to wells (total 13սL).
6. Put lid on. Run at 100V, until about 1/2 way down the plate.
7. Transport in tubberware to image.
8. Lanes that appeared to “run” well after imaging were inoculated on 8/16/2010.

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