Homo sapiens - 2p21 (SPG4): PCR primers for exon amplification and sequencing

Exon	PCR product size (bp)	PCR program	PCR Primers	Sequence primers	PCR1	PCR2	Sequencing
1	1048	0	gSPAex1c	GTGAGCCGAACTGCACATTG	x
			gSPAex1m	CAAAGTCGACAGCTACAGTGC	x
	975	0	gSPAex1d	GGAACTGTAGTTGAGTGGGA		x	x
			gSPAex1n	AGATGAGGCTCCGACCTAC		x	x
2	624	3	gSPAex2a	AATGCCACACTTGTAATCTC	x
			gSPAex2m	TGTGAATATATCATAATTTGGG	x		x
			gSPAex2b	TACAGCAGTTCTCATGATG			x
3	812	1	gSPAex3a	GACCAAATTGGTGCATGCATG	x
			gSPAex3m	ACATTTCCAATACATCCCAC	x		x
4	379	3	gSPAex4a	ATTTGTCATTTCACATGCAC	x
			gSPAex4m	TTAGAATGACTATACCTGAC	x
			gSPAex4n	TCAGGTTAAGTAAGACTC			x
5	830	4	gSPAex5a	TTCCTATCTACCTAGTGAC	x
			gSPAex5m	TTTTATAGCAAGTTGCCCTG	x
			gSPAex5b	CCTATGAAGATCCTGGTAC			x
6	484	3	gSPAex6a	TGTCATGATTCTAACAAGGG	x		x
			gSPAex6m	TCTATTTCACTCCTGACATG	x
7	420	2	gSPAex7a	GTCATAGGGCTTAGGCTTC	x
			gSPAex7m	ATCATACTACCCACTTTTCC	x		x
8	647	3	gSPAex8a	TGTTTGGGAAGATGCTACTG	x		x
			gSPAex8m	CTACTGAAGATAACGTACATG	x
9	1268	1	gSPAex9a	CATTGATTGCCATGTATTGG	x
			gSPAex9m	AGAAGGCCAGAAATACTCAG	x
			gSPAex9b	GTACTTAAATCGGTAAATATGG			x
10 11 12	1081	4	gSPAex10a	CTCAAGTCTTAGGAATGCAG	x
			gSPAex10m	GCTATGTGAGTCATCTGAG			x
			gSPAex11a	CTCAGATGACTCACATAGC			x
			gSPAex12m	CTTTACTAGACTAATTCTCCTG	x		x
13	1361	4	gSPAex13a	CAGATTCAAGAAGACAGATC	x
			gSPAex13m	GCAATAATTCACCACACTTG	x
			gSPAex13n	GGTAGTTCTTGTTTCTGCTC			x
14	1003	4	gSPAex14a	CAAGTGTGGTGAATTATTGC	x
			gSPAex14m	GAGCTGAAAAGTATTCAGC	x
			gSPAex14n	TGCAAAGGACATAGCCAGTG			x
15	1076	1	gSPAex15a	AGCCTCTGGAGATAGTATGC	x
			gSPAex15m	CTAGAACAGGGGTCACAGTC	x
			gSPAex15n	TTGGACTTCTTAAACTTC			x
16	1404	4	gSPAex16a	GCAGTATGCAAGAAATTGAAC	x
			gSPAex16m	GGCCTGTAATTTTCTTCTG	x
			gSPAex16b	GTACTGAATAGATACATGTAG			x
17	445	3	gSPAex17a	GTGTAGCAGATCAACATAG	x		x
			gSPAex17m	CATCTTCAAGTTTGGTGCAC	x

PCR Programs

All reactions where performed in 50 µl final, with :

100 ng genomic DNA
50 pmol each primer
250 µM dNTPs
1x Takara mix
1.5 u Takara Taq polymerase (Takara La Taq, Shuzo)

Hot start PCR procedure was used, Taq polymerase was added at 94°C after a 5 min 96°C denaturation step.
Samples where then processed through 30 cycles of :

Program #	Denaturation	Annealing	Elongation
0	Advantage GC genomic PCR kit (Clonetech)
1	40s 94°C	50s 60°C	1min 72°C
2		50s 58°C	40s 72°C
3		50s 55°C	40s 72°C
4		50s 55°C	1min 72°C

A final elongation step of 10 min 72°C was applied.

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