Hosuk:LabNotes/2015-1-25
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CA12k_Nov2014 : New Padlock Probes[edit]
Concentration[edit]
- CA12k_Nov2014 that Matt has produced
- V4 primers - CA12k_Nov2014_Contig
- V7 primers - CA12k_Nov2014_Const
- Produced at 2015-01-09
- V4 : 10.7 ng/ul --> 10.7 ng/ul / (150nt*325Da/nt + 79Da) = 219nM (38ul)
- V7 : 6.2 ng/ul --> 6.2 ng/ul / (150nt*325Da/nt + 79Da) = 127nM (18ul)
- I’ve used 18uL of V4, and 18uL of V7 for the sample at 1/22
- --> V4 : 103.7nM, V7 : 127nM in 100uL Ampligase Mix
- --> 18.67 nM of V4, 22.86 nM of V7 in 100uL mix
- Let’s fix the final concentration of Padlock probes to 100nM in 100uL Ampligase Mix
- Produced at 2015-01-12
- V7 : 28 ng/ul --> 28 ng/ul / (150nt*325Da/nt + 79Da) = 573nM (18ul)
- Produced at 2015-01-15
- V4 : 65.3 ng/ul --> 65.3 ng/ul / (150nt*325Da/nt + 79Da) = 1337nM (38ul)
- V7 : 39.2 ng/ul --> 39.2 ng/ul / (150nt*325Da/nt + 79Da) = 803nM (18ul)
CA12k_Nov2014 - Test aminoallyl dUTP[edit]
- I want to test anaspec’s a-dUTP that I’ve just got, and comparing to the result without a-dUTP
- Sample : PGP1F, P13, cultured since 1/13/15
- (1) 1/24 : Fix sample
- Take out the dishes from CERC to the lab, and fix them with 10% Formaldehyde for 15min at 37C.
- Wash with PBS 3 times, then incubate with 70% EtOH for 5 min, then 0.1N HCL for 1 min.
- (2) 1/24 : Do RT ~ 15hr.
- S1, S2 : with a-dUTP, 4mM, anaspec
- S3, S4 : with a-dUTP, 2mM, life tech(?)
- S5, S6 : no a-dUTP
- (3) 1/25 : BS(PEG)9 (opened at 1/11/15), and Tris pH 8.0
- S1, S2, S3, S4 only
- (4) RNaseH : all samples
- S1, S3, S5 : Run Ampligase with 100nM (final conc.) of CA12k_Nov2014_V4
- S2, S4, S6 : Run 1st Rolony generation
- (5-1) Ampligase Mix
- Padlock probe : 2015-01-15 V4 : 1337nM (38ul)
- -->100nM in final conc. --> 7.5uL in 100uL Ampligse Mix
- Buffer : 10 uL
- Ampligase (5U/uL) : 10 uL --> 0.5U/uL in final conc.
- Padlock Probe (V4, 1/15/15) : 7.5 uL
- H2O : 72.5 uL
- -->Ampligase : 24 hr.
- (5-2) Circligase II for 1st Rolony generation
- Mix
- Buffer : 10 uL
- MnCl2 : 5 uL
- Betaine : 20 uL
- CircLigase II : 2 uL
- H2O : 63 uL
- -->Run : 2 hr.
- Mix
- (6-1) Run RCA : FISSEQ_RCA – S2, S4, S6 (1st rolony generation)
- S4, S6 : 2mM a-dUTP, S2 : 4mM a-dUTP
- (6-2) Run RCA : FISSEQ_ppRCA – S1, S3, S5 (V4 PP)
- S3, S5 : 2mM a-dUTP, S1 : 4mM a-dUTP
- RCA mix
- Buffer : 10 uL
- dNTP : 2 uL
- a-dUTP : 2 uL for 4mM, 4uL for 2mM
- Phi29 : 2 uL
- H2O : 84 uL for 4mM, 82 uL for 2mM