Daniel:Notebook/ComboLock/2016-8-5

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Revision as of 17:08, 5 August 2016 by >Djacobse (→‎Oligonucleotide Design)
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Positive Control Amplicon Production

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In order to test the latch-padlock binding better, I am going to use a positive control oligonucleotide. The oligo is designed to contain only the parts of the C probes (and antibody oligos) that bind to the latch and padlock, as well as barcodes. The oligos will also have biotin, which means I'll have a good method for pulldown.

Oligonucleotide Design

First thing is to design the oligos. The following table indicates the created oligonucleotide sequences. The LatchX1, C1 amplicon and C2 amplicon were ordered from IDT. The reason for the additional latch is to remove the UMI on the normal latch sequences. Since the UMI is a degenerate sequence I can't design an oligo to bind to it. Therefore I replaced the UMI on the normal latch sequences with a second barcode for the LatchX1 probe. There are two amplicons because IDT doesn't allow you to attach a biotin to ultramers (>60 bp).

Name Sequence Reverse Complement Length (bp) Components
primer2 ACGGCGGACCTCGCACGG CCGTGCGAGGTCCGCCGT 18  
primer4 CCGTGGACGGTCGCGTTC GAACGCGACCGTCCACGG 18  
primer6 CGGCGATGCGTGATCGGG CCCGATCACGCATCGCCG 18  
primer12 CCCGCCCAGTAACCGGCG CGCCGGTTACTGGGCGGG 18  
LatchX1 Barcode CATTTAGTATACGGGC GCCCGTATACTAAATG 16  
Latch 8 barcode TATTTGTA TACAAATA 8  
Latch 9 barcode GCGCCTAC GTAGGCGC 8  
Total Amplicon ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTT CGCCCGTATACTAAATGCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG CGCCGGTTACTGGGCGGGGTAGGCGCCCCGATCACGCATCGCC GCATTTAGTATACGGGCGAACGCGACCGTCCACGGTACAAATACCGTGCGAGGTCCGCCGT 104 Primer2-Latch8BC-Primer4-LatchX1BCRevComp-Primer6-Latch9BC-Primer12
C1 Amplicon ACGGCGGACCTCGCACGGTATTTGTACCGTGGACGGTCGCGTTCACTAAATG CATTTAGTGAACGCGACCGTCCACGGTACAAATACCGTGCGAGGTCCGCCGT 52  
C2 Amplicon GCCCGTATCGGCGATGCGTGATCGGGGCGCCTACCCCGCCCAGTAACCGGCG CGCCGGTTACTGGGCGGGGTAGGCGCCCCGATCACGCATCGCCGATACGGGC 52  
Latch X1 CAAGATCATAAGAGTTCGTTCGTCCACCACACAGCGACGAGACCG CGGTCTCGTCGCTGTGTGGTGGACGAACGAACTCTTATGATCTTG 45 primer6RC-bclp0001-bclp0002-primer4RC

Ligation Reaction

Since the amplicon is in pieces right now, I need to ligate the C1 and C2 amplicons together.

Theory

Protocol