Chapter 2: Materials and Methods
2.8 DNA sequencing
47 2.7.5 PCR amplification of Tn-DNA junctions
Genomic DNA fragments were amplified to enrich Tn junction sites by PCR amplification using a primer with Tn homology and a primer with homology to the adapter sequence (see Table 2.4). For the PCR, 8 reactions were prepared for each sample. Briefly, for each PCR, 100 ng of DNA was added into 2.5 µl of DMSO, 5 µl of 10× PCR buffer, 5 µl of 2.5 mM dNTPs, 0.5 µl Taq DNA polymerase, 1.5 µl of 10 µM adapter primer and 1.5 µl of 10 µM Tn primer. Water was added to each reaction to bring it up to a volume of 30 μl. PCR parameters were 95ºC for 10 min; 20 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 45 sec; and 72°C for 5 min. After PCR, 8 reactions were pooled for each sample. The amplified DNA was visualized on a 2% agarose gel run at 60 V for 140 min. Smears of amplified DNA in the 400- 600 bp range were excised, placed in the -20°C freezer for 30 min and then gel extracted with the Qiagen Gel Extraction kit and eluted into 50 μl water.
2.7.6 Hemi-nested PCR
The staggered primers were mixed together in equal concentration prior to being added to the PCR mix (Table 2.4). For hemi-nested PCR, 4 μl of amplified DNA product from the first round of PCR was mixed with 2 µl of 1 µM Sol-mar mix, 2 µl of 1 µM Sol-ap-tag mix, 2µl of 10× PCR buffer, 2 µl of 2.5 mM dNTPs, 0.2 µl of Taq polymerase and water was added up to final volume of 20 µl. PCR parameters were 95°C for 5 min; 10 cycles of 95°C for 30 sec, 58°C for 30 sec and 72°C for 45 sec; and 72ºC for 5 min. Amplified DNA was purified using the QIAquick PCR purification kit as per the manufacturer’s instructions and and eluted into 50 μl EB buffer. The DNA concentration was measured by Qubit.
48
Table 2.4: Primers and adapters used for TnSeq. The long Sol_AP1 primers recognize the adapter portion of the sequence whereas Sol_Mar recognizes the Tn (Long et al., 2015).
Primer name Sequence Function
Adapter 1 5′-TACCACGACCA-NH2 Barcoded adapter
that ligate to genomic DNA after fragmentation Adapter 2 5′ATGATGGCCGGTGGATTTGTGNNANNANNNTGG
TCGTGGTAT JEL_AP1
(Adapter primer)
5′-ATGATGGCCGGTGGATTTGTG Amplifies the
sequence between barcoded genomic DNA fragment and inserted Tn.
T7
(Tn primer)
5′-TAATACGACTCACTATAGGGTCTAGAG
1.Sol_AP 1_ tagged 930
A CAAGCAGAAGACGGCATACGAGATTGTTCCGAGT GACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTC AATGATGGCCGGTGGATTTGTG
Multiplex sequencing adapter. Long primers which inserts specific sequence (in bold) allowing
identification of samples after multiplex sequencing.
B CAAGCAGAAGACGGCATACGAGATTGTTCCGAGT GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGT CCATGATGGCCGGTGGATTTGTG
C CAAGCAGAAGACGGCATACGAGATTGTTCCGAGT GACTGGAGTTCAGACGTGTGCTCTTCCGATCTACA GTCCCATGATGGCCGGTGGATTTGTG
D CAAGCAGAAGACGGCATACGAGATTGTTCCGAGT GACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAG TGGATGATGGCCGGTGGATTTGTG
2.Sol_AP 1_tagged_
949
A CAA GCA GAA GAC GGC ATA CGA GAT TTC CGG AGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT GTC AAT GAT GGC CGG TGG ATT TGT G B CAA GCA GAA GAC GGC ATA CGA GAT TTC CGG
AGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT CGT CCA TGA TGG CCG GTG GAT TTG TG
C CAA GCA GAA GAC GGC ATA CGA GAT TTC CGG AGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT ACA GTC CCA TGA TGG CCG GTG GAT TTG TG
D CAA GCA GAA GAC GGC ATA CGA GAT TTC CGG AGG TGA CTG GAG TTC AGA CGT GTG CTC TTC
49
CGA TCT TAG TGG ATG ATG GCC GGT GGA TTT GTG
3.Sol_AP 1_tagged_
651
A CAA GCA GAA GAC GGC ATA CGA GAT GCC GAT GTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT GTC AAT GAT GGC CGG TGG ATT TGT G B CAA GCA GAA GAC GGC ATA CGA GAT GCC GAT
GTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT CGT CCA TGA TGG CCG GTG GAT TTG TG
C CAA GCA GAA GAC GGC ATA CGA GAT GCC GAT GTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT ACA GTC CCA TGA TGG CCG GTG GAT TTG TG
D CAA GCA GAA GAC GGC ATA CGA GAT GCC GAT GTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT TAG TGG ATG ATG GCC GGT GGA TTT GTG
4.Sol_AP 1_tagged_
373
A CAA GCA GAA GAC GGC ATA CGA GAT CAT GAT CGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT GTC AAT GAT GGC CGG TGG ATT TGT G B CAA GCA GAA GAC GGC ATA CGA GAT CAT GAT
CGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT CGT CCA TGA TGG CCG GTG GAT TTG TG
C CAA GCA GAA GAC GGC ATA CGA GAT CAT GAT CGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT ACA GTC CCA TGA TGG CCG GTG GAT TTG TG
D CAA GCA GAA GAC GGC ATA CGA GAT CAT GAT CGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT TAG TGG ATG ATG GCC GGT GGA TTT GTG
5.Sol_AP 1_tagged_
473
A CAA GCA GAA GAC GGC ATA CGA GAT CGC GCG GTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT GTC AAT GAT GGC CGG TGG ATT TGT G B CAA GCA GAA GAC GGC ATA CGA GAT CGC GCG
GTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT CGT CCA TGA TGG CCG GTG GAT TTG TG
50
C CAA GCA GAA GAC GGC ATA CGA GAT CGC GCG GTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT ACA GTC CCA TGA TGG CCG GTG GAT TTG TG
D CAA GCA GAA GAC GGC ATA CGA GAT CGC GCG GTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT TAG TGG ATG ATG GCC GGT GGA TTT GTG
6.Sol_AP 1_tagged_
100
A CAA GCA GAA GAC GGC ATA CGA GAT ACA CGA TCG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT GTC AAT GAT GGC CGG TGG ATT TGT G B CAA GCA GAA GAC GGC ATA CGA GAT ACA CGA
TCG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT CGT CCA TGA TGG CCG GTG GAT TTG TG
C CAA GCA GAA GAC GGC ATA CGA GAT ACA CGA TCG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT ACA GTC CCA TGA TGG CCG GTG GAT TTG TG
D CAA GCA GAA GAC GGC ATA CGA GAT ACA CGA TCG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT TAG TGG ATG ATG GCC GGT GGA TTT GTG
7.Sol_AP 1_tagged_
57
A CAA GCA GAA GAC GGC ATA CGA GAT AAG TAG AGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT GTC AAT GAT GGC CGG TGG ATT TGT G B CAA GCA GAA GAC GGC ATA CGA GAT AAG TAG
AGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT CGT CCA TGA TGG CCG GTG GAT TTG TG
C CAA GCA GAA GAC GGC ATA CGA GAT AAG TAG AGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT ACA GTC CCA TGA TGG CCG GTG GAT TTG TG
D CAA GCA GAA GAC GGC ATA CGA GAT AAG TAG AGG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT TAG TGG ATG ATG GCC GGT GGA TTT GTG
8.Sol_AP 1_tagged_
598
A CAA GCA GAA GAC GGC ATA CGA GAT GAG ATC TTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT GTC AAT GAT GGC CGG TGG ATT TGT G
51
B CAA GCA GAA GAC GGC ATA CGA GAT GAG ATC TTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT CGT CCA TGA TGG CCG GTG GAT TTG TG
C CAA GCA GAA GAC GGC ATA CGA GAT GAG ATC TTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT ACA GTC CCA TGA TGG CCG GTG GAT TTG TG
D CAA GCA GAA GAC GGC ATA CGA GAT GAG ATC TTG TGA CTG GAG TTC AGA CGT GTG CTC TTC CGA TCT TAG TGG ATG ATG GCC GGT GGA TTT GTG
9.Sol_
Mar
A AATGATACGGCGACCACCGAGATCTACACTCTTT CCCTACACGACGCTCTTCCGATCTCGGGGACTTAT CAGCCAACC
Common
sequencing primer which recognizes the Tn.
B AATGATACGGCGACCACCGAGATCTACACTCTTT CCCTACACGACGCTCTTCCGATCTTCGGGGACTTA TCAGCCAACC
C AATGATACGGCGACCACCGAGATCTACACTCTTT CCCTACACGACGCTCTTCCGATCTGATACGGGGA CTTATCAGCCAACC
D AATGATACGGCGACCACCGAGATCTACACTCTTT CCCTACACGACGCTCTTCCGATCTATCTACGGGGA CTTATCAGCCAACC