<< Manually Evaluate Primer Quality

In this step we will check the primer list of left & right primers generated by the Bio-Informatics tool Primer-3.

Primer3 Output

PRIMER PICKING RESULTS FOR hap RSRS

No mispriming library specified
Using 1-based sequence positions
SEQUENCE SIZE: 16569
INCLUDED REGION SIZE: 650

Please take some time to look through this list of Primers recommended by the Bio-Informatics tool Primer3.
Compare to the two panels of recommendations above. Let's start looking at the left and right primer first by itself not as a pair. We will do that later.
Please consider length, melting temperature, GC content and the 3'-ends of some of these primers.


Once you have a list of your mutations, please select a left and right primer from this list paying attention to the starting position of these primers and the length.
The Left primer's "start" position should be lower than the base pair position for the first expected mutation minus the length of primer.
The Right primer's "start" position should be higher than the base pair position for the last expected mutation plus the length of primer.
What is an indication that any of these RSRS specific primers are universal primer? e.g. it can amplify DNA of any haplogroup?

Action: Once you have settled on a primer pair move on to next question in worksheet Section 4.2.

                        start  len      tm     gc%  any_th  3'_th hairpin       seq 
1	LEFT_PRIMER	15972	21	58.73	47.62	0	0	0	TAACTCCACCATTAGCACCCA
2	LEFT_PRIMER	15973	20	58.93	50	0	0	0	AACTCCACCATTAGCACCCA
3	LEFT_PRIMER	15974	20	58.93	50	0	0	0	ACTCCACCATTAGCACCCAA
4	LEFT_PRIMER	15975	21	58.91	52.38	0	0	0	CTCCACCATTAGCACCCAAAG
5	LEFT_PRIMER	15975	20	57.79	50	0	0	0	CTCCACCATTAGCACCCAAA
6	LEFT_PRIMER	15976	20	57.79	50	0	0	0	TCCACCATTAGCACCCAAAG
7	LEFT_PRIMER	15977	20	59.47	55	0	0	0	CCACCATTAGCACCCAAAGC
8	LEFT_PRIMER	16029	20	57.85	50	0	0	0	TCATGGGGAAGCAGATTTGG
9	LEFT_PRIMER	16030	20	58.88	55	0	0	0	CATGGGGAAGCAGATTTGGG
10	LEFT_PRIMER	16031	20	59.29	50	0	0	0	ATGGGGAAGCAGATTTGGGT
11	LEFT_PRIMER	16031	21	59.07	47.62	0	0	0	ATGGGGAAGCAGATTTGGGTA
12	LEFT_PRIMER	16032	20	58.62	50	0	0	0	TGGGGAAGCAGATTTGGGTA
13	LEFT_PRIMER	16032	19	58.83	52.63	0	0	0	TGGGGAAGCAGATTTGGGT
14	LEFT_PRIMER	16033	20	58.24	55	0	0	0	GGGGAAGCAGATTTGGGTAC
15	LEFT_PRIMER	16064	20	57.73	50	0	0	0	TGACTcACCCATCAACAACC
16	LEFT_PRIMER	16065	20	58.84	55	0	0	0	GACTcACCCATCAACAACCG
17	LEFT_PRIMER	16067	19	59.12	57.89	0	0	0	CTcACCCATCAACAACCGC
18	LEFT_PRIMER	16069	20	58.25	50	0	0	0	cACCCATCAACAACCGCTAT
19	LEFT_PRIMER	16070	20	58.25	50	0	0	0	ACCCATCAACAACCGCTATG
20	LEFT_PRIMER	16071	20	58.25	50	0	0	0	CCCATCAACAACCGCTATGT
21	LEFT_PRIMER	16093	20	57.98	50	0	0	0	TTCGTACATTACTGCCAGCC
22	LEFT_PRIMER	16094	20	59.1	50	0	0	0	TCGTACATTACTGCCAGCCA
23	LEFT_PRIMER	16099	20	59.26	55	0	0	0	CATTACTGCCAGCCACCATG
24	LEFT_PRIMER	16101	20	59.6	50	0	0	0	TTACTGCCAGCCACCATGAA
25	LEFT_PRIMER	16101	19	58.93	52.63	0	0	0	TTACTGCCAGCCACCATGA
26	LEFT_PRIMER	16102	20	59.37	50	0	0	0	TACTGCCAGCCACCATGAAT
27	LEFT_PRIMER	16102	19	58.93	52.63	0	0	0	TACTGCCAGCCACCATGAA
28	LEFT_PRIMER	16102	21	59.16	47.62	0	0	0	TACTGCCAGCCACCATGAATA
29	LEFT_PRIMER	16103	20	59.37	50	0	0	0	ACTGCCAGCCACCATGAATA
30	LEFT_PRIMER	16104	20	58.01	50	0	0	0	CTGCCAGCCACCATGAATAT
31	LEFT_PRIMER	16105	21	58.97	47.62	0	0	0	TGCCAGCCACCATGAATATTG
32	LEFT_PRIMER	16166	21	58.74	47.62	0	0	0	ACCCAAtCCAcATCAAAACcC
33	LEFT_PRIMER	16231	21	58.78	47.62	0	0	0	TCACACATCAaCtGCAACTCC
34	LEFT_PRIMER	16232	21	59.05	47.62	0	0	0	CACACATCAaCtGCAACTCCA
35	LEFT_PRIMER	16238	20	58.97	50	0	0	0	TCAaCtGCAACTCCAAAGCC
36	LEFT_PRIMER	16239	20	59.25	50	0	0	0	CAaCtGCAACTCCAAAGCCA
37	LEFT_PRIMER	16240	20	59.25	50	0	0	0	AaCtGCAACTCCAAAGCCAC
38	LEFT_PRIMER	16278	21	59.15	47.62	0	0	0	tCAACAAACCTAcCcacCCTt
39	LEFT_PRIMER	16346	20	57.97	55	0	0	0	GTCAAATCCcTTCTCGtCCC
40	LEFT_PRIMER	16347	20	59.09	55	0	0	0	TCAAATCCcTTCTCGtCCCC
41	LEFT_PRIMER	16348	20	59.38	55	0	0	0	CAAATCCcTTCTCGtCCCCA
42	LEFT_PRIMER	16349	20	58.4	50	0	0	0	AAATCCcTTCTCGtCCCCAT
43	LEFT_PRIMER	16350	20	59.16	55	0	0	0	AATCCcTTCTCGtCCCCATG
44	LEFT_PRIMER	16395	19	59.02	57.89	0	0	0	CTTGACCACCATCCTCCGT
45	LEFT_PRIMER	16410	21	58.78	47.62	0	0	0	CCGTGAAATCAATATCCCGCA
46	LEFT_PRIMER	16436	19	59.27	63.16	0	0	0	GTGCTACTCTCCTCGCTCC
47	LEFT_PRIMER	16524	20	57.96	50	0	0	0	AAGCCTAAATAGCCCACACG
48	LEFT_PRIMER	16525	20	59.09	50	0	0	0	AGCCTAAATAGCCCACACGT
49	LEFT_PRIMER	16526	20	58.25	50	0	0	0	GCCTAAATAGCCCACACGTT
50	LEFT_PRIMER	16536	20	57.79	50	0	0	0	CCCACACGTTCCCCTTAAAT

                        start  len      tm     gc%  any_th  3'_th hairpin       seq 
1	RIGHT_PRIMER	16555	20	57.79	50	0	0	0	ATTTAAGGGGAACGTGTGGG
2	RIGHT_PRIMER	16454	19	59.27	63.16	0	0	0	GGAGCGAGGAGAGTAGCAC
3	RIGHT_PRIMER	16413	19	59.02	57.89	0	0	0	ACGGAGGATGGTGGTCAAG
4	RIGHT_PRIMER	16369	20	59.16	55	0	0	0	CATGGGGaCGAGAAgGGATT
5	RIGHT_PRIMER	16368	20	58.4	50	0	0	0	ATGGGGaCGAGAAgGGATTT
6	RIGHT_PRIMER	16367	20	59.38	55	0	0	0	TGGGGaCGAGAAgGGATTTG
7	RIGHT_PRIMER	16366	20	59.09	55	0	0	0	GGGGaCGAGAAgGGATTTGA
8	RIGHT_PRIMER	16365	20	57.97	55	0	0	0	GGGaCGAGAAgGGATTTGAC
9	RIGHT_PRIMER	16298	21	59.15	47.62	0	0	0	aAGGgtgGgTAGGTTTGTTGa
10	RIGHT_PRIMER	16297	20	58.47	50	0	0	0	AGGgtgGgTAGGTTTGTTGa
11	RIGHT_PRIMER	16259	20	59.25	50	0	0	0	GTGGCTTTGGAGTTGCaGtT
12	RIGHT_PRIMER	16258	20	59.25	50	0	0	0	TGGCTTTGGAGTTGCaGtTG
13	RIGHT_PRIMER	16257	20	58.97	50	0	0	0	GGCTTTGGAGTTGCaGtTGA
14	RIGHT_PRIMER	16252	21	59.05	47.62	0	0	0	TGGAGTTGCaGtTGATGTGTG
15	RIGHT_PRIMER	16251	21	58.78	47.62	0	0	0	GGAGTTGCaGtTGATGTGTGA
16	RIGHT_PRIMER	16191	20	59.59	55	0	0	0	GGgGaGgGTTTTGATgTGGa
17	RIGHT_PRIMER	16186	21	58.74	47.62	0	0	0	GgGTTTTGATgTGGaTTGGGT
18	RIGHT_PRIMER	16126	21	58.97	47.62	0	0	0	aCAATATTCATGGTGGCTGGC
19	RIGHT_PRIMER	16125	21	58.97	47.62	0	0	0	CAATATTCATGGTGGCTGGCA
20	RIGHT_PRIMER	16123	20	58.01	50	10.94	10.94	0	ATATTCATGGTGGCTGGCAG
21	RIGHT_PRIMER	16122	20	59.37	50	17.16	0	0	TATTCATGGTGGCTGGCAGT
22	RIGHT_PRIMER	16122	21	59.16	47.62	17.16	2.72	0	TATTCATGGTGGCTGGCAGTA
23	RIGHT_PRIMER	16121	20	59.37	50	17.16	2.72	0	ATTCATGGTGGCTGGCAGTA
24	RIGHT_PRIMER	16120	20	59.6	50	17.16	1.38	0	TTCATGGTGGCTGGCAGTAA
25	RIGHT_PRIMER	16120	19	58.93	52.63	17.16	2.72	0	TTCATGGTGGCTGGCAGTA
26	RIGHT_PRIMER	16119	19	58.93	52.63	17.16	1.38	0	TCATGGTGGCTGGCAGTAA
27	RIGHT_PRIMER	16115	20	58.62	55	17.16	6.4	0	GGTGGCTGGCAGTAATGTAC
28	RIGHT_PRIMER	16114	20	58.99	55	17.16	0	0	GTGGCTGGCAGTAATGTACG
29	RIGHT_PRIMER	16113	20	59.1	50	17.16	0	0	TGGCTGGCAGTAATGTACGA
30	RIGHT_PRIMER	16112	20	57.98	50	17.16	0	0	GGCTGGCAGTAATGTACGAA
31	RIGHT_PRIMER	16089	20	58.25	50	0	0	0	CATAGCGGTTGTTGATGGGT
32	RIGHT_PRIMER	16088	20	58.25	50	0	0	0	ATAGCGGTTGTTGATGGGTg
33	RIGHT_PRIMER	16087	20	59.31	50	0	0	0	TAGCGGTTGTTGATGGGTgA
34	RIGHT_PRIMER	16085	19	59.12	57.89	0	0	0	GCGGTTGTTGATGGGTgAG
35	RIGHT_PRIMER	16084	20	58.84	55	0	0	0	CGGTTGTTGATGGGTgAGTC
36	RIGHT_PRIMER	16083	20	57.73	50	0	0	0	GGTTGTTGATGGGTgAGTCA
37	RIGHT_PRIMER	16053	20	58.24	55	0	0	0	GGTACCCAAATCTGCTTCCC
38	RIGHT_PRIMER	16052	20	58.24	55	0	0	0	GTACCCAAATCTGCTTCCCC
39	RIGHT_PRIMER	16051	20	58.62	50	0	0	0	TACCCAAATCTGCTTCCCCA
40	RIGHT_PRIMER	16051	21	59.07	47.62	0	0	0	TACCCAAATCTGCTTCCCCAT
41	RIGHT_PRIMER	16050	20	59.29	50	0	0	0	ACCCAAATCTGCTTCCCCAT
42	RIGHT_PRIMER	16050	19	58.83	52.63	0	0	0	ACCCAAATCTGCTTCCCCA
43	RIGHT_PRIMER	16049	20	58.88	55	0	0	0	CCCAAATCTGCTTCCCCATG
44	RIGHT_PRIMER	16048	20	57.85	50	0	0	0	CCAAATCTGCTTCCCCATGA
45	RIGHT_PRIMER	15996	20	59.47	55	0	0	0	GCTTTGGGTGCTAATGGTGG
46	RIGHT_PRIMER	15995	21	58.91	52.38	0	0	0	CTTTGGGTGCTAATGGTGGAG
47	RIGHT_PRIMER	15995	20	57.79	50	0	0	0	CTTTGGGTGCTAATGGTGGA
48	RIGHT_PRIMER	15994	20	57.79	50	0	0	0	TTTGGGTGCTAATGGTGGAG
49	RIGHT_PRIMER	15993	20	58.93	50	0	0	0	TTGGGTGCTAATGGTGGAGT
50	RIGHT_PRIMER	15992	20	58.93	50	0	0	0	TGGGTGCTAATGGTGGAGTT

Statistics
         con   too    in    in   not          no    tm    tm   high  high  high        high      
         sid  many   tar  excl    ok   bad    GC   too   too any_th 3'_th hair-  poly   end      
        ered    Ns   get   reg   reg   GC% clamp   low  high  compl compl   pin     X  stab    ok
Left    3323     0     0     0     0   244     0  1681   299      0     0   424   114     0   485
Right   3323     0     0     0     0   244     0  1681   299      0     0   455   114     0   467
Pair Stats:
considered 0, ok 0
libprimer3 release 2.3.7


(primer3_results.cgi release 4.0.0)