Nested PCR
- The initial DNA extract template is subjected to amplification with the template-specific primers LR0R and JHY-LSU-369rc for 30 cycles. The number of cycles is lower than usual to prevent various biases resulting from excessive amplification.
- PCR reactions are not cleaned up, to avoid loss of DNA (we used to do it, but it is probably not necessary).
- 5μl of each amplicon is run on a gel to assess failures/successes. You may not see very much on your gel! Even faint band should be used. When band is invisible, I wouldn’t use it even though there may still be some amplicon – it was probably not a good unbiased amplification.
- A 1-μl aliquot of each successful amplicon is used as a subsequent PCR template and subjected to a 20-cycle amplification with the fusion primers.
- The secondary (final) amplicons are cleaned up with AmPure DNA cleanup kit.
- The DNA concentration in each cleaned PCR product is measured using the PicoGreen-based Quant-iT kit.
- Products are pooled in equimolar proportions to create an amplicon library at a final concentration of 10 ng μl−1.
- The pooled amplicon library is cleaned up using UltraClean PCR clean-up kit and submitted to ICBR.
- When submitting to ICBR, make sure you specify that we need raw output, non-demultiplexed fastq data.
The LSU fusion primers
LR0R –> JH-LSU-369rc: From 5’ of the first primer site to the 3’ end of the reverse primer: 400bp. The most variable region (positions): 100-240
Primer construction
To prepare an order, simply concatenate. You need one Forward, and as many Reverse as you need barcodes. We order form IDT, which only does synthesis at 100nM for oligos this long. I have negotiated this synthesis for the price of 25nM synthesis, so do purchases through me. Specify that you want them in the “Lab Ready” suspension.
Forward primer:
P5 AATGATACGGCGACCACCGA
Linker GATCT
SBS3 ACACTCTTTCCCTACACGACGCTCTTCCGATCT
LR0R ACCCGCTGAACTTAAGC
Reverse primer:
P7 CAAGCAGAAGACGGCATACGA
Linker GAT
Barcode xxxxxxx
SBS12 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
JH-LS-369rc CTTCCCTTTCAACAATTTCAC
Barcodes:
We already have the JH-LS-369rc fusion primers with these barcodes :
1 AAGGTGG
8 GCTGAGG
15 ACAGCGG
23 CATGGTG
31 TCGGTTG
35 CGAGATG
41 TGTGCTG
45 CTAGGAG
51 TATGTAG
56 GTGGAAG
60 TAATCAG
64 TCTGGCG
70 CCAGTCG
75 CTGTACG
78 GATGCCG
80 ACGTGGT
85 TCTGTGT
93 TAAGAGT
98 GTTGCGT
103 ATTGGTT
109 CTGGATT
114 TAGGCTT
120 GGTGGAT
127 CAAGTAT
134 TCGTAAT
138 CCGGCAT
142 AATTGCT
146 ACGGTCT
150 GAGGACT
155 TTAGCCT
158 CATTGGA
167 GATCTGA
176 GCTGGTA
181 AGTGACA
189 TTCAGGC
202 GACGGTC
Other available barcodes:
2 CTTGTGG
3 TGAGTGG
4 CAATTGG
5 TTCTTGG
6 GGTATGG
7 GTGCTGG
9 ATCGAGG
10 GAGTAGG
11 TGTTAGG
12 AGGAAGG
13 TCGCAGG
14 AATCAGG
16 TACGCGG
17 ATGTCGG
18 GGATCGG
19 CAGACGG
20 GTCACGG
21 CGTCCGG
22 TTACCGG
24 TAGTGTG
25 AGATGTG
26 CTCTGTG
27 ACTAGTG
28 TGCAGTG
29 ATGCGTG
30 GCACGTG
32 GTAGTTG
33 AGCGTTG
34 CTGATTG
36 ACGTATG
37 TTATATG
38 GTTAATG
39 AACAATG
40 CAGCATG
42 GCCGCTG
43 AATTCTG
44 CCATCTG
46 CCTTGAG
47 GGAAGAG
48 CACAGAG
49 TGGCGAG
50 GATCGAG
52 GCGTTAG
53 ATATTAG
54 TCCATAG
55 AGTCTAG
57 TGCGAAG
58 CTCCAAG
59 ATTGCAG
61 AGCTCAG
62 GCTACAG
63 AAGCCAG
65 GTCGGCG
66 AAGAGCG
67 CTTAGCG
68 CCGCGCG
69 TAACGCG
71 TGGTTCG
72 AGAATCG
73 CATCTCG
74 ATCCTCG
76 GCATACG
77 GGCAACG
79 TCGACCG
81 CTATGGT
82 CGTAGGT
83 GACAGGT
84 AGACGGT
86 ATAGTGT
87 TAGTTGT
88 GCATTGT
89 AGCTTGT
90 CCGATGT
91 AATATGT
92 CTCCTGT
94 CACTAGT
95 GTGAAGT
96 TCCAAGT
97 CGGCAGT
99 CGAGCGT
100 CCTTCGT
101 TGGACGT
102 GAGCCGT
104 TCAGGTT
105 GTGTGTT
106 AGGAGTT
107 CCTCGTT
108 TACCGTT
110 GGCGATT
111 GCTTATT
112 TATAATT
113 AGTCATT
115 CGGTCTT
116 ATCTCTT
117 GCGACTT
118 CTTACTT
119 GTACCTT
121 AACGGAT
122 GAATGAT
123 CGCTGAT
124 TTGAGAT
125 ACAAGAT
126 GCGCGAT
128 GCCGTAT
129 CTGTTAT
130 TGATTAT
131 GAGATAT
132 GTTCTAT
133 AGCAGGA
135 GTCTAAT
136 CATCAAT
137 GGACAAT
139 AGTACAT
140 TACACAT
141 TCTCCAT
143 TTCTGCT
144 GCTAGCT
145 TGAAGCT
147 CGTGTCT
148 TCACTCT
149 GGCCTCT
151 AGATACT
152 CTCAACT
153 TTGCACT
154 AACCACT
156 GGTTCCT
157 ATGACCT
159 GCCTGGA
160 CTGAGGA
161 TAGCGGA
162 ACTCGGA
163 GTACGGA
164 CGCGTGA
165 TTAATGA
166 AGGCTGA
168 CCACTGA
169 CTAGAGA
170 GACGAGA
171 CCGTAGA
172 TTCCAGA
173 AATGCGA
174 TGCTCGA
175 TCTACGA
177 TCGAGTA
178 GTCAGTA
179 CGGCGTA
180 ACAGTTA
182 TATTACA
183 CGGAACA
184 CTTCACA
185 CTGGCCA
186 ACCGCCA
187 TCATCCA
188 TTCACCA
190 GTCGTGC
191 CAGCTGC
192 TGTCTGC
193 ACGGAGC
194 CGTGAGC
195 TTGTAGC
196 GGCTAGC
197 GTTCAGC
198 AGCGCGC
199 GATTCGC
200 CTCTCGC
201 GCACCGC
203 ACCTGTC
204 GGTAGTC
205 AATCGTC
