medium: 10%FBS in 1x AA, warm up for 1 hour before use
pass 1 to 4 in petri dish when crowded (twice a week):
vacuum out old medium, add 4mL trypsin-EDTA, incubate 3 minutes until cells float to top, add 4mL medium.
centrifuge 5 minutes, vacuum out supernatant, add 4mL medium to cells, mix thoroughly.
Add 1mL to each of 4 new petri dishes.
add medium to make total of 5mL solution if transferring to small petri dishes, 25mL if transferring to large petri dishes.
Thursday, August 12, 2010
Thursday, July 29, 2010
Wednesday, July 7, 2010
Tuesday, July 6, 2010
Thursday, July 1, 2010
Wednesday, June 30, 2010
MSR1 IHC revised
1. incubate in three washes of xylene for 3 minutes each
2. incubate in two washes of 100% ethanol for 2 minutes each
3. incubate in one wash of 95% ethanol for 2 minutes
4. incubate in one wash of 70% ethanol for 2 minutes
6. Wash twice in dH2O for 2 minutes each
7. Rinse
Antigen unmasking:
1. heat sections in unmasking solution (1mm EDTA pH 8.0) at full power (microwave) for 2 minutes to boil, then heat at low power (PL-1) for 15 minutes.
Staining
*** premake PBST stock!
1. Wash in dH2O twice for 5 minutes each, add directly onto hot solution.
2. Using blocker marker, draw boundary around tissue section on slides. Incubate in 3% Hydrogen Peroxide for 10 minutes. (denatures endogenous enzymes)
3. Wash in dH2O twice for 5 minutes each
4. Washing in PBST (PBS+0.1% Tween 20) for 5 minutes
5. Block each section with 100-400microliter blocking buffer for one hour at RT (blocking buffer: PBST+5% rabbit normal serum).
6. Remove blocking solution, apply diluted primary antibody to slides, incubate at least 1 hour at room temperature (MSR1 Ab: cat #MAB1797 from R&D, dilute1:150 in blocking buffer).
7. Remove antibody solution and wash in (1/200) PBST three times for 5 minutes each.
8. Apply 100-400microliter diluted biotinylated secondary antibody to each section (dilute 1:200 in blocking buffer), incubate at RT for 1 hour.
9. Wash slides in PBST three times for 5 minutes each.
10. Apply 100-400microliter ABC reagent to each section, incubate at RT for 30 minutes.
11. Wash slides in PBST three times for 5 minutes each.
12. Add 100-400 microliter DAB to each section and monitor staining closely.
13. As soon as the sections develop, immerse slides in dH2O.
14. counterstain in hematoxylin (10 seconds)
15. Rinse in dH2O until water is clear
16. Dehydrate: 10 sec in 95% ethanol --> 10 sec in 95% ethanol --> 10 sec in 100% ethanol --> 10 sec in 100% ethanol --> 10 sec in xylene --> 10 sec in xylene
17. Mount with Cytoseal Xylene Mounting medium (From VMR, cat number 48212-196).
Monday, June 28, 2010
MSR1 IHC Procedure
Deparaffinization/Rehydration:
1. incubate in three washes of xylene for 3 minutes each
2. incubate in two washes of 100% ethanol for 2 minutes each
3. incubate in one wash of 95% ethanol for 2 minutes
4. incubate in one wash of 70% ethanol for 2 minutes
5. incubate in one wash of 50% ethanol for 2 minutes
6. Wash twice in dH2O for 2 minutes each
Antigen unmasking:
1. heat sections in unmasking solution (citrate pH=6, vector labs) at full power for 2 minutes to boil, then heat at low power (PL-1) for 10 minutes.
2. Cool slides on bench top for 30 minutes
Staining
1. Wash in dH2O twice for 5 minutes each.
2. Incubate in 3% Hydrogen Peroxide for 10 minutes.
3. Wash in dH2O twice for 5 minutes each
4. Washing in PBST (PBS+0.1% Tween 20) for 5 minutes
5. Block each section with 100-400microliter blocking buffer for one hour at RT (blocking buffer: PBST+5% rabbit normal serum).
6. Remove blocking solution, apply diluted primary antibody to slides, incubate overnight at $ degrees Celsius (MSR1 Ab: cat #MAB1797 from R&D, dilute1:150 in blocking buffer).
7. Remove antibody solution and wash in PBST three times for 5 minutes each.
8. Apply 100-400microliter diluted biotinylated secondary antibody to each section (dilute 1:200 in blocking buffer), incubate at RT for 1 hour.
9. Wash slides in PBST three times for 5 minutes each.
10. Apply 100-400microliter ABC reagent to each section, incubate at RT for 30 minutes.
11. Wash slides in PBST three times for 5 minutes each.
12. Add 100-400microliter DAB to each section and monitor staining closely.
13. As soon as the sections develop, immerse slides in dH2O.
14. counterstain in hematoxylin
15. Rinse in dH2O until water is clear
16. Dehydrate: 10 sec in 95% ethanol --> 10 sec in 95% ethanol --> 10 sec in 100% ethanol --> 10 sec in 100% ethanol --> 10 sec in xylene --> 10 sec in xylene
17. Mount with Cytoseal Xylene Mounting medium (From VMR, cat number 48212-196).
1. incubate in three washes of xylene for 3 minutes each
2. incubate in two washes of 100% ethanol for 2 minutes each
3. incubate in one wash of 95% ethanol for 2 minutes
4. incubate in one wash of 70% ethanol for 2 minutes
5. incubate in one wash of 50% ethanol for 2 minutes
6. Wash twice in dH2O for 2 minutes each
Antigen unmasking:
1. heat sections in unmasking solution (citrate pH=6, vector labs) at full power for 2 minutes to boil, then heat at low power (PL-1) for 10 minutes.
2. Cool slides on bench top for 30 minutes
Staining
1. Wash in dH2O twice for 5 minutes each.
2. Incubate in 3% Hydrogen Peroxide for 10 minutes.
3. Wash in dH2O twice for 5 minutes each
4. Washing in PBST (PBS+0.1% Tween 20) for 5 minutes
5. Block each section with 100-400microliter blocking buffer for one hour at RT (blocking buffer: PBST+5% rabbit normal serum).
6. Remove blocking solution, apply diluted primary antibody to slides, incubate overnight at $ degrees Celsius (MSR1 Ab: cat #MAB1797 from R&D, dilute1:150 in blocking buffer).
7. Remove antibody solution and wash in PBST three times for 5 minutes each.
8. Apply 100-400microliter diluted biotinylated secondary antibody to each section (dilute 1:200 in blocking buffer), incubate at RT for 1 hour.
9. Wash slides in PBST three times for 5 minutes each.
10. Apply 100-400microliter ABC reagent to each section, incubate at RT for 30 minutes.
11. Wash slides in PBST three times for 5 minutes each.
12. Add 100-400microliter DAB to each section and monitor staining closely.
13. As soon as the sections develop, immerse slides in dH2O.
14. counterstain in hematoxylin
15. Rinse in dH2O until water is clear
16. Dehydrate: 10 sec in 95% ethanol --> 10 sec in 95% ethanol --> 10 sec in 100% ethanol --> 10 sec in 100% ethanol --> 10 sec in xylene --> 10 sec in xylene
17. Mount with Cytoseal Xylene Mounting medium (From VMR, cat number 48212-196).
Tuesday, June 15, 2010
Friday, June 11, 2010
Wednesday, June 2, 2010
Tuesday, June 1, 2010
Thursday, May 27, 2010
cDNA synthesis
Background Info:
http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm
mix:
water
reaction mix
RNA
enzyme (keep on ice)
add RNA
tap it
PCR
http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm
mix:
water
reaction mix
RNA
enzyme (keep on ice)
add RNA
tap it
PCR
Protein Extraction
http://www.piercenet.com/browse.cfm?fldID=FA97D803-6953-48E4-A7BD-6947D35FE83B
Basic Protocol:
Mix:
Lysis Buffer
Protease Inhibitor (comes in pellet --> vortex with buffer)
Phosphorase Inhibitor
add to pulverized tumor samples
homogenize while keeping tube on ice
spin down using vortex
Basic Protocol:
Mix:
Lysis Buffer
Protease Inhibitor (comes in pellet --> vortex with buffer)
Phosphorase Inhibitor
add to pulverized tumor samples
homogenize while keeping tube on ice
spin down using vortex
Wednesday, May 26, 2010
Tuesday, May 25, 2010
Monday, May 24, 2010
BLAST Algorithm and Substitution Matrix
http://en.wikipedia.org/wiki/BLAST#Algorithm
http://en.wikipedia.org/wiki/Substitution_matrix
http://en.wikipedia.org/wiki/Substitution_matrix
Sequence Alignment
http://en.wikipedia.org/wiki/Sequence_alignment
*know global vs local alignment, various programs: FASTA, genebank, dot matrix-hits on the main diagonal determines sequence similarity
*know global vs local alignment, various programs: FASTA, genebank, dot matrix-hits on the main diagonal determines sequence similarity
Poisson Distribution
http://en.wikipedia.org/wiki/Poisson_distribution
http://bioinfo.mbb.yale.edu/course/classes/c4/c4-p1.html
"Having introduced the idea of approximating the binomial distribution with two distributions, each of which is applicable in a different regime of the value of p, lets consider the case where p is small (p0.1). First, let us perform the substitution l =np. The binomial distribution then becomes,
(7)
Now, consider the case where n grows to infinity and p shrinks to zero. Hopefully you appreciate the utility of the substitution that we made above, since we can force n to grow and p to shrink such that l =np remains constant. This is nice since nothing in the above expression will "blow up" for large n and/or small p. In this limit we get,
(8) for k=0,1,2,3,...
This expression is the Poisson distribution, and is useful in the situations where the probability of an occurrence is small and the number of "trials" (n) is large. For example, we might consider the probability of k adverse reactions to a test drug in a given sample of the population or the probability of registering k complaints about a particular product in a 1-hour period or the probability of finding k point mutations in a given stretch of nucleotides. Though the Poisson distribution is essential to application and you will doubtless see it again, we will leave it now to discuss the other binomial-approximating continuous distribution. "
http://bioinfo.mbb.yale.edu/course/classes/c4/c4-p1.html
"Having introduced the idea of approximating the binomial distribution with two distributions, each of which is applicable in a different regime of the value of p, lets consider the case where p is small (p0.1). First, let us perform the substitution l =np. The binomial distribution then becomes,
(7)
Now, consider the case where n grows to infinity and p shrinks to zero. Hopefully you appreciate the utility of the substitution that we made above, since we can force n to grow and p to shrink such that l =np remains constant. This is nice since nothing in the above expression will "blow up" for large n and/or small p. In this limit we get,
(8) for k=0,1,2,3,...
This expression is the Poisson distribution, and is useful in the situations where the probability of an occurrence is small and the number of "trials" (n) is large. For example, we might consider the probability of k adverse reactions to a test drug in a given sample of the population or the probability of registering k complaints about a particular product in a 1-hour period or the probability of finding k point mutations in a given stretch of nucleotides. Though the Poisson distribution is essential to application and you will doubtless see it again, we will leave it now to discuss the other binomial-approximating continuous distribution. "
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