Summer Research
went in today... split the bes21 cells..
did a gel for msx and mpri and 1341
1341 didnt show up.... started transformation for plasmid again
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
Worked until 8PM
did gel in the morning, and carried out experiment... different time points, sonification, luciferase assay
ahh so much stuff
Wednesday (yesterday)
after plasmid maxi prep tried to find the concentration of DNA with a spectrometer... low readings... going to do a gel tomorrow
