Summer Research
yesterday, split cells and changed media
today - started transfection
1. Transformation of MSX2-Lux and MPRI plasmids in bacteria
take ~ 50ul of competant bacteria cells, thaw on ice and then add ~ 5ng of the above plasmids to cells and incubate on ice for 30 min. Heat shock at 37*C for 30 seconds and replace on ice for 2 min. Add 1ml of LB broth to transformation mix and shake for 1hr at 37*C. Then take ~50ul and spread on agar plateto grow overnight
2. pick colony and culture in 2-3ml of LB with ampicilin (50ug/ml) for ~ 6 hrs and then transfer 200ul to 250ml LB (50ug/ml AMP) for overnight culture.
3. purify plasmid using qiagen kit
BMPRI 1ug/ul 1000ul water to dilute to 1ng/ul
MSX 0.8ug/ul 800ul water to dilute to 1ng/ul
1341 0.44ug/ul 440ul water to dilute to 1ng/ul
LB
950ml water
bacto tryptone 10g
bacto yeast extract 5g
NaCL 10g
agar plate
250ml LB + 15g/L bactor agar
250ml of LB - autoclave and let stand at room temp till ~50*C add 250ul ampicillin to adjust to 50ug/ml final concentration
Then mswirl to mix amp with media, immediately pour media into plate ~ 10ml each, use fisher 100mm dishes
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afternoon splitted mda-mb-435 cells
got bes21 cells from nitrogen tank cs yang 6th canister 2nd to last box
replaced media for MCF-7 cells
washed and lysed plates 2 3 4 5
24hr gccg 24hr 3TP 24hr cmyc
put egcg, catalase and and h2O2 into wells
incubating for 5 and 24 hours
plan to add cAMP around 4pm to mdr
Did trasnfection today
Design
1. GCCG
2. GCCG + catalase (50units)
3. GCCG + EGCG (20um)
4. GCCG + EGCG + catalase
5. GCCG + H2O2
6. GCCG H2O2 + catalase
time points 5 hr and 24 hr
6 x 2 time pts x 3 triplicates = 36 wells
13. 3TP
14. 3TP + catalase
15. 3TP + EGCG
16. 3TP + EGCG + catalase
17. 3TP + H2O2
18. 3TP + H2O2 + catalase
one time point x triplicates = 18 wells
36 + 18 = 54 wells
3 plates
Calculations
for plates 1 and 2
4ml media + 254 ul GCCG DNA
4ml of media + 40 ul lipofectamine
for plate 3
28.6 ul 3TP DNA + 2ml media
20ml lipofectamine + 2ml media
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Design for MDR cells
01. c-Myc
02. c-Myc + cAMP
03. c-Myc + RIAZ
04. c-Myc + RiAZ +cAMP
05. c-Myc + R1(a)
06. c-Myc + R1(a) + cAMP
07. c-Myc + R1(a)mut
08. c-Myc + R1(a)mut + cAMP
09. c-Myc + R1(a) + RIAZ
10. c-Myc + RI(a) + RIAZ + cAMP
11. c-Myc + R1(a)mut + RIAZ
12. c-Myc + R1(a)mut + RIAZ + cAMP
came in on sunday (8/3/03), plated bes, and mdr cells, and splitted cells
BES21 cells:
1. resuspended cells in ~ 7 ml and plate 4x10^4 cells per well
2. take 0.5ml and save for stock
54 wells ~ 60 wells 2.4ml in 30ml + 30ml media
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MDA-MB-435 cells
1. trypsinize cells with 1.5ml of trypsin incubate cells at 37*C for ~ 3-4 min. Shake flask gently by tapping edge of flask against corner of hand to dislodge cells. Resuspend cells in 4.5ml of RPMI media to yield a total of 6ml of cell suspenion (~1x10^6 cells/ml)
2. plate ~4x10^4 cells per well as below:
1) myc-luc
2) myc-luc + cAMP
3) myc-luc + RIAZ
4) myc-luc + RIAZ + cAMP
5) myc-luc + R1a
6) myc-luc + R1a + caMP
7) myc-luc + R1a + RIAZ
8) myc-luc + R1a + RIAZ + cAMP
9) myc-luc + R1a-mut
10 ) myc-luc + R1a-mut + cAMP
11) myc-luc + R1a-mut + RIAZ
12) myc-luc + R1a-mut + RIAZ + cAMP
12x3=36 wells
~40 wells 1.6ml in 40ml
2. Take ~ 200ul MDA-MB-435 cells and plate in T25 for stock. Add ~9ml of media to flask for future culture
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MCF-7
1. Split cellsby trypsinizing with 1.5ml trypsin and incubate at 37*C for ~ 3-4min
2. Tap to dislodge cells and then resuspend with 4.5ml of media
3. Take ~ 200ul of MCF-7 cells for stock in T25