Sequencing

DNA sequencing is performed on PCR products isolated from a DNA agarose electrophoresis gel to ensure that all impurities are eliminated.

3.7.1. Purification of the PCR Product

The PCR product is purified from the agarose using the QIAquick Gel Extraction Kit.

1. Excise DNA bands from gel.

2. Transfer the bands into individual 1.5-mL sterile microfuge tubes and add 300 ||L buffer QG.

3. Incubate at 50°C for 10 min and vortex each for 2-3 min to completely dissolve the gel.

4. Add 100 |L 100% isopropanol and vortex to mix.

5. Place the QIAquick spin column in a 2-mL collection tube and apply the sample.

6. Centrifuge for 1 min.

7. Discard flow through and replace the column in the same tube.

Primers Used for Conventional and Real Time Reverse Transcriptase Polymerase Chain Reaction to Analyze Transcript Levels

Forward

Reverse

Annealing

primer

primer

temperature

Product

Gene

(5'—>3')

(5'—3')

(°C)

size (bp)

D-Loop

cga aag gac aag aga aat aag g

ctg taa agt ttt aag ttt tat gcg

55

181

COX I

tca taa tcg gag gct ttg gc

caa cct caa cac cac ctt ct

55

421

COX II

atc atc cta gtc ctc atc gc

aga cgt ctt gca ctc atg ag

55

280

COX III

cgc gat gta aca cga gaa ag

tca gag tac ttc gag tct cc

55

379

NRF1

gga gtg atg tcc gca cag aa

cgc tgt taa gcg cca tag tg

58

584

Tfam

tat caa gat gct tat agg gc

act cct cag cac cat att tt

55

441

NRF2a

tag acc tca cca cac tca ac

gtg acc aaa cgg ttc aac tc

58

627

NRF2ß

gag ctc cct tta cta cag ac

aac tgt ggt gtt gca gca tg

58

462

PolG

cat tgg aca tcc aga tgc tc

cct gat atg agc tcg gtc aa

58

679

TFB1M

tct gca atg ttc gac aca tc

acc tat ata aga agc tcc ac

49

501

TFB2M

aga agc agt tcc ttg gac ag

agt ggt cta tta cag tgg cg

57

463

B-ACTIN

tgg cac cac acc ttc tac aat gag c

gca cag ctt ctc ctt aat gtc acg c

55

400

Oct-4

cga cca tct gcc gct ttg ag

ccc cct gtc ccc cat tcc ta

60

241

a-MHC

gga gga gca agc caa cac caa

gca gtg agg ttc ccg tgg ca

59

179

ANF

tag gga cag act gca aga gg

cga gga agt cac cat caa acc ac

59

577

Nkx2.5

tgg cta cag ctg cac tgc cg

gga tcc atg cag cgt gga c

60

165

Rex-1

gcg tac gca aat taa agt cca ga

cag cat cct aaa cag ctc gca gaa t

56

306

SOX-2

ccc ccg gcg gca ata gca

tcg gcg ccg ggg aga tac at

55

448

FGF-4

cta caa cgc cta cga gtc cta ca

gtt gca cca gaa aag tca gag ttg

55

NRF 2a

TFAM

NRF 2a

Fig. 1. Regulation of mtDNA transcription and replication factors. Differentiating and undifferentiating hESCs exhibit low levels of mtDNA transcription and replication factors, TFAM and NRF2a, as do hESC-derived cardiomyocytes. However, migratory hESCs have higher levels of NRF2a. Adult cells, human heart and TOV cells, consistently express high levels of all three transcription factors. U, undifferentiated and M, migratory hESCs i.e., those cells migrating from the central colony; EBM, ethidium bromide-treated M; EBU, ethidium bromide-treated U; Rho+, hESCs cultured in the same media as ethidium bromide-treated cells; C, hESC-derived cardiomyocytes; heart, adult human heart; TOV, an ovarian tumor cell line; L, 100 bp DNA ladder (Invitrogen); neg, negative control reaction. For further details see ref. 46.

Fig. 1. Regulation of mtDNA transcription and replication factors. Differentiating and undifferentiating hESCs exhibit low levels of mtDNA transcription and replication factors, TFAM and NRF2a, as do hESC-derived cardiomyocytes. However, migratory hESCs have higher levels of NRF2a. Adult cells, human heart and TOV cells, consistently express high levels of all three transcription factors. U, undifferentiated and M, migratory hESCs i.e., those cells migrating from the central colony; EBM, ethidium bromide-treated M; EBU, ethidium bromide-treated U; Rho+, hESCs cultured in the same media as ethidium bromide-treated cells; C, hESC-derived cardiomyocytes; heart, adult human heart; TOV, an ovarian tumor cell line; L, 100 bp DNA ladder (Invitrogen); neg, negative control reaction. For further details see ref. 46.

8. Add 0.5 mL of buffer QG and centrifuge for 1 min.

9. Wash the DNA with 0.75 mL of buffer PE containing ethanol and centrifuge 1 min. 10. Discard flow-through, then centrifuge for 1 min and place column into a new

1.5-mL microcentrifuge tube.

11. Elute the DNA in 50 ||L autoclaved sterile ddH2O and centrifuge for 1 min.

3.7.2. Cycle Sequencing: Preparing the Reaction

1. For each PCR reaction, add 3.2 pmol of forward primer to 20 ng of purified DNA product, obtained from the purified PCR, in a final reaction volume of 10 |L.

2. Apply the total sample volume in one of the wells in the 96-well reaction plate.

3. Add 8.0 |L Terminator Ready Reaction Mix consisting of 2 |L of Reaction Premix and 6 |L of BigDye Sequencing Buffer.

4. Mix well and spin briefly.

3.7.3. Cycle Sequencing: PCR Conditions Used

1. Perform 25 cycles of the following parameters: 96°C for 10 s; 50°C for 5 s; 60°C for 4 min; and 4°C (infinity).

2. Following the cycling reaction, spin down the contents of the tubes in a microcentrifuge and proceed to purify the extension products.

3.7.4. Purification of Extension Products

1. Pipet entire contents of the reaction into a 1.5-mL microcentrifuge tube.

3. Add 64 |L of nondenatured 95% ethanol.

4. Close the tube and vortex briefly.

5. Incubate at room temperature for 15 min (see Note 11).

6. Centrifuge the tubes for 20 min at maximum speed and proceed immediately to step 7.

7. Carefully aspirate the supernatant with a separate tip for each sample and discard (see Notes 12 and 13).

8. Add 250 |L of 70% ethanol and vortex briefly. Repeat.

9. Spin for 10 min at maximum speed.

10. Aspirate the supernatant carefully as in step 7.

11. Dry the samples in a vacuum centrifuge for 10-15 min or to dryness (see Notes 14 'and 15).

Was this article helpful?

0 0

Post a comment