Chris:LabNotes/FateMapping/Calendar/2014/2014-10-16
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Padlock Probe Production (designed 9/22)[edit]
Background[edit]
- Previously, I designed a new set of probes that were filtered such that the capture arms would very specifically map to the desired target (see <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2014/2014-9-22>)
- Because of the limited size of the array probe production, we were only able to order 8,045 probes (all 127bp in length, with v6 primers used to amplify)
- As listed on Dr. Zhang's wiki <http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/MONOD/2014-8-4>, the v6 primer sequences are (G*T*CATATCGGTCACTGTU//5Phos/GGGTAGTGTGTATCCTG). For reference, the v6 primer sequences based on the primer order form are (G*G*GTCATATCGGTCACTGTU//5Phos/CACGGGTAGTGTGTATCCTG).
- The details on the CA tube are:
Custom Array CA-1127 90k_oligos_30Sept2014 69.89ng/ul 80ul -> 5.59ug 10/14/14
Procedure[edit]
- I used the same protocol that Noi's been using for probe capture
Expansion PCR[edit]
- Considering the length of the probes are 127bp, I calculated that the concentration in the tube is 1667.62nM
- Mastermix recipe (want to run 2 rxn's)
Component 1x (ul) Oligo Mix 5.997 KAPA FAST MM (2x) 50 100uM AP1V61U 0.40 100uM AP2V6 0.40 H2O 43.203
- Noi noticed that with her probe set, there was an undesirable strong smear below 100bp. I ran a gel (as shown below) in order to verify the expansion PCR worked:
File:Cw ZhangLab 2 2014-10-16 17hr 58min pp production exp pcr.jpg
- Unfortunately, I also see a strong smear below 100bp, when I would expect a strong band at 127bp