Team:OU Norman/Project/Notebook/erin

4/10/14 3A Assembly of mlsR and ter

Calculations of DNA to use:

• 500 ng (upstream part) x 470.6 ng/ μl = 1.06 = 1 μl mlsR #24
• 500 ng ter 6d#16 (downstream part) x 194.2 ng/ μl = 2.57= 2.5 μl ter 6d#16
• 500 ng ter 4p #10 (downstream part) x 299.8 ng/ ul= 1.67 = 2 μl ter 4p #10
• 500 ng pSB1K3 x 175.2 ng/ μl = 2.85= 3 μl pSB1K3

Digest

Order of reagents added:

1. H2O
2. BSA (used to coat inside of tube)
3. 10 x buffer
4. DNA
5. Enzyme

Total reaction volume was 50 mu;L.
Reactions were placed in 42°C water bath for 45min
Reactions placed on heat block at 80°C for 15 min to heat kill enzymes

**Used Thermo Science buffer for all reactions even though some enzymes were from different company. If digest fails, try different combos of buffer.

Ligation

Order of reagents added:

1. H2O
2. 10x buffer
3. DNA
4. Enzyme

Total reaction volume was 40μl
Reactions were incubated on bench top for 20min

Transformation

1. 4μl of ligation was added to 100 μl aliquot of TOP10 competent cells (x5)
2. Reaction kept on ice for 30 min
3. Reaction heat shocked in 42°C water bath for 60 sec
4. Reaction put on ice for 5 min
5. 600 μl Psi broth added to each reaction
6. Tubes incubated with shaking at 200 rpm for 2 hours at 37°C
7. Undiluted (1:1) cells were plated (70 μl)
8. 1:10 dilution was made with remainder of cells (10 μl transformation with 90 μl Psi) and all 100 μl was plated onto plates with kanamycin
9. Plates incubated at 37°C overnight

RESULTS IN DIFFERENT NOTEBOOK

7/17/14-7/18/14 Competent cells (E. coli DH5α with pIKM1)

7/17: 10 ml LB 2X YT inoculated with single colony ~3pm
7/18: Competent cell protocol started ~11pm, Used 1ml cells instead of 1.5 ml

7/21/14-7/26/14 Transformation of E. coli DH5α PIKM1 with P1#4 and P2#2

P1(Ori + pPtb- + mlsR + 4p + pSB1C3) = 210.1ng/ μl
P2 (Ori + pPtb- + mlsR + 6d + pSB1C3) = 198.8 ng/ μl

7/21:

1. 100 μl of competent cells (E. coli DH5α pIKM1) plus 2.5 μl (~500ng) plasmid
2. Reaction kept on ice for 30 min
3. Reaction heat shocked in 42°C water bath for 60 sec
4. Reaction put on ice for 5 min
5. 600 μl Psi broth added to each reaction
6. Tubes incubated with shaking at 200 rpm for 2 hours at 37°C
7. Undiluted (1:1) cells were plated (50 μl )
8. Plates (LB 2xYT + Chloramphenicol) incubated at 37°C overnight

7/22-26: No colonies
Are the competent cells good?
Need a new protocol for cell to accommodate 2 plasmids?
Try electrotransformation?




7/29/14 Electrotransformation of E. coli DH5α pIKM1 with P1#4 and P2#2




Preparation of electrocompetent DH5α pIKM1 cells:



1. Grew up overnight of E. coli DH5α pIKM1 from single colony in ~30 mL of LB 2x YT at 200 rpm and 37°C
2. Brought LB 2xYT closer to room temp
3. Made 1ml overnight into 100mL fresh LB 2xYT dilution
4. Incubated at 37°C with 200 rpm until OD 0.5 was reached
5. 1.5 ml culture aliquoted into each of 10 tubes
6. Tubes spun at 14000 rpm for 8 min
7. Supernatant removed



REALIZED THAT CELLS WERE GROWN OVERNIGHTIN LB + SALT AND NEED TO BE GROWN IN LB-SALT FOR ELECTROPORATION-EXPREIMENT STOPPED




8/1/14 Wrong Plasmid




It turns out that Dr. Wawrik gave us E. coli containing the wrong plasmid. It should be E. coli DH5α pAN1.




E. coli DH5α pAN1 was streaked out onto a LB+chloramphenicol plate for overnight growth at 37°C.




This still does not address the question of why our transformation did not work. Streaked competent cells make on 7/18 onto LB +kanamycin plate and LB without antibiotics plate to see if compent pIKM1 cells made still have pIKM1 plasmid (and thus chloramphenicol resistance) and are viable to determine if need different chemical competent cell protocol.




8/3/14 Viability of Competent Cells




Chemically competent cells plated on LB 2xYT and LB 2xYT + Kanamycin both grew significantly, suggesting that our competent cell protocol maintained cell viability and didn’t kick the pIKM1 plasmid out. Perhaps the protocol was just done wrong, fresh media needs to be used, or a protocol needs to be adapted to allow the cell to accommodate 2 plasmids. Electrotransformation will be attempted.




8/4/14 Electrotransformation of E. coli DH5α pAN1 with P1#4 and P2#2




Preparation of electrocompetent DH5α pIKM1 cells:



1. Started overnight culture in saltless LB 2xYT at 9:08pm on 8/3/14
1. 25 mL 2xYT
2. Colonies were really close together
2. Made dilution
3. 1. 1.25 mL overnight into 50mL saltless 2xYT (x2)
4. Incubated 2 bottles of dilutions at 37°C with 200 rpm until ~OD 0.5 was reached
1. Bottle 1 OD .66
2. Bottle 2 OD .59
5. Bottles transferred to ice to chill
6. 15 1mL tubes from each bottle were prepared
7. Tubes spun at 14,000 rpm for 8min
8. Supernatant removed
10. Resusupended in 500 ul cold ~90% glycerol
1. Glycerol was prepared incorrectly
11. Spun 14000 rpm for 3 min
12. Removed supernatant
13. Resuspended in 200ul cold 10% glycerol
14. Spun 14000 rpm 3 min
15. Removed supernatant
16. Resuspended in 100μl 10% cold glycerol
17. Put cells in -80°C armor beads to freeze
18. Stored in -80°C



Electrotransformation (E. coli DH5α pIKM1 with P1 and P2)



1. Combine in separate cuvettes:
1. 2.5 ul P1 + 100ul cells (bottle 1) P1 #1
2. 2.5 ul P1 + 100 ul cells (bottle 2) P1 #2
3. 2.5 ul P2 + 100 ul cells (bottle 1) P2 #1
4. 2.5 ul P2 + 100 ul cells (bottle 2) P2 #2
2. Let sit 5 min
3. Set electroporator to 2500V
4. Dumped Cell/plasmid mixture into special elctroporator cuvette
5. Hit run
6. Took out of electroporator, immediately pipetted in 600ul of Psi broth, and placed on ice.
7. Took from cuvette, placed in tube, incubated at 37°C with 200 rpm for 2 hours.
8. Plated 50 ul of 1:100 and 1:1000 dillutions onto LB2xYT plates + chloramphenicol



Electroporator readings




Sample	Actual Voltage	Time (ms)
Control	2400	4.8
P2 #1	640	0.7
P2 #2	2350	3.9
P1 #2	2370	4.3
P1 #1	1990	1.6




P2 #1 may not have worked.




8/5/14 Results Electrotransformation E. coli DH5α pAN1 with P1#4 and P2#2

Colonies grew on plates
Libraries made by Sam
Not sure if both plasmids there because both have chloramphenicol resistance. Should grow on erythromycin plates, but not sure if will work since mlsR is behind Gram positive promoter.
Clolony PCR will be performed to test for presence of plasmid. Gel will be run to see if both of our plasmids are there.




8/6/14 Colony PCR to verify presence of P1 and P2 in E. coli DH5α pAN1




P2 #2 pAN1 = 12 reactions
P1 #1 pAN1= 14 reactions
26 reactions total
In 1.5 ml tube:



• 450 ul Master mix
• 446 ul PCR H2O
• 2μl Fwd Primer
• 2μl Rvrs Primer

900 μl total




30 μl aliquotted into each PCR tube
*Barely touch each colony with tooth pick and suspend in tube




P1 #1 +pAN1




Tube #	Colony #
1	1
2	2
3	3
4	4
5	5
6	6
7	7
8	8
9	9
10	10
11	11
12	12
13	13
14	17




P2 #2 + pAN 1




Tube #	Colony #
15	1
16	3
17	4
18	5
19	7
20	8
21	9
22	10
23	11
24	12
25	17




Results in other notebook.




8/8/14 Gel of Colony PCR



• 5μl dye
• 2μl dye
• 117 volts



Large gel (TOP)




Lane	Sample
1	Ladder
2	P1
3	P2
4	pAN1
5	Tube 1
6	Tube 2
7	Tube 3
8	Tube 4
9	Tube 5
10	Tube 6
11	Tube 7
12	Tube 8
13	Tube 9
14	Tube 10
15	Tube 11
16	Ladder




Large gel (BOTTOM)




Lane	Sample
1	Ladder
2	P1
3	P2
4	pAN1
5	Tube 12
6	Tube 13
7	Tube 14
8	Tube 15
9	Tube 16
10	Tube 17
11	Tube 18
12	Tube 19
13	Tube 20
14	Tube 21
15	Tube 22
16	Ladder




Small Gel




Lane	Sample
1	Ladder
2	P1
3	P2
4	pAN1
5	Tube 23
6	Tube 24
7	Tube 25
8	Dye




Appear to have light bands in some of the wells on the top portion of the large gel, however, these bands don’t correspond to the size of either plasmid. In preparing the gel, some dye without florescent agent was used by accident, so there is possibility that plasmid is there, we just can’t visualize it.




9/8/ 14 Digest of plasmid extraction from transformed E. coli DH5α pAN1 with P1 or P2

Sample	EcoR1 (&mu:l)	10x buffer (&mu:l)	PCR water (&mu:l)	DNA (&mu:l)
pAN1 + P1#2	1	2	1	8.96	7.04
pAN1 + P1#1	1	2	1	10.48	5.52
pAN1 + P2#1	1	2	1	12.06	3.94
pAN1 + P2#2	1	2	1	9.94	6.06
pAN1	1	2	1	13.12	2.88
P1	1	2	1	14.66	1.34
P2	1	2	1	14.70	1.30




Order of reagents added:



1. water
2. BSA
3. 10 x buffer
4. DNA
5. Enzyme



Heat bath (42°C) for 45 min
Heat block (80°C) for 15 min

Results in other notebook.