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1. cDNA library inserts can be amplified using primers that flank the
inserts. T3/T7 and M13 sequences are frequently found to be flanking
inserts in cDNA libraries. They have been widely used for PCR
amplification of cDNA library inserts.
2. Sequences of T3 and T7 primers:
T3: 5'-CGA AAT TAA CCC TCA CTA AAG GGA
(Melting temperature 57 °C, nearest neighbor method)
T7: 5'-GTA ATA CGA CTA CTA TAG GG
(Melting temperature 48 °C, nearest neighbor method)
Sequences of M13 primers:
M13 Forward: 5'-GTT TTC CCA GTC ACG ACG TTG
(Melting temperature 59 °C, nearest neighbor method)
M13 Reverse: 5'-TGA GCG GAT AAC AAT TTC ACA CAG
(Melting temperature 58 °C, nearest neighbor method)
See also Common PCR and sequencing primers.
3. Single clones can be amplified using the
PCR amplification of single clones protocol. Or clones can be
grown in 96 wells and directly amplified by PCR as described below.
4. cDNA clones are inoculated into 150 ml of
LB with antibiotics in 96-well microtiter plates, and grown over night
in a 37 °C incubator. Place plates in a zip-lock bag with a wet paper tower.
5. The over night cultures or frozen stocks are diluted 1:20 in ddH2 and 2
ml of the
diluted culture is used per PCR reaction.
6. Make a master mix of PCR reaction buffer.
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Single Reaction
|
96 reactions
|
| 10X PCR Reaction Buffer |
5 ml |
500 ml |
| Forward primer (10 mM, 10 pmol/ml ) |
1 ml |
100 ml |
| Reverse primer (10 mM, 10 pmol/ml ) |
1 ml |
100 ml |
| 25 mM MgCl2 |
4 ml |
40 ml |
| 10 mM dNTP (mix of 10 mM dATP, dCTP, dGTP, dTTP each) |
1 ml |
100 ml |
| PCR DNA Polymerase (1 units/ml) |
1 ml |
100 ml |
| ddH2O |
35 ml |
3500 ml |
| Total volume |
48 ml |
4800 ml |
7. For each clone, add 48 ml of master mix to
2 ml of culture supernatant in 96 well PCR palte.
8. Amplify in a thermocycler using the following cycling protocol:
| Step 1 |
Denaturation 94 °C |
12 minutes |
| Step 2 |
Denaturation 94 °C |
30 seconds |
| Step 3 |
Annealing 55 °C (M13 primers) |
30 seconds |
| Step 4 |
Extension 72 °C |
45 seconds |
| Step 5 |
Repeat step 2-4 29 times |
|
| Step 6 |
Extension 72 °C |
45 seconds |
| Step 7 |
Storage 4 °C |
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