Colony PCR
Screening the Transposition of Your Gene of Interest into the Viral Genome
1. Prepare selective agar plates as in the electroporation and transposition procedure.
2. Label the appropriate number of 0.5 ml microcentrifuges tubes. Place on ice.
3. To each tube add:5 µl of 10X PCR buffer1 µl of 10 mM dNTP mix1.5 µl of 50 mM MgCl22.5 µl of primer mix (1.25 each 10 µM stock)0.5 µl Taq Polymerase (2.5 U)Add DDW to a final volume of 50 µl
4. Mix the contents of the tube by gently tapping the tube.
5. Pick a colony from the plate into the reaction mix.
6. Perform PCR as follows:a) 93oC for 3 minb) 94oC for 45 secc) 55oC for 45 secd) 72oc for 5 minperform steps b-d x40 cycles.
7. Prepare 0.7% agarose gel in 1xTAE containing 0.5 µg/ml ethidium bromide
8. Load 24µl of each sample on gel (prepare the sample as follows: 20 µl of PCR reaction + 4 µl of 6x loading buffer). Run gel at 100 mA.
9. The expected results from the PCR are as follows: Bacmid alone ~300 bp Bacmid transposed with pFastBac1 ~2300 bp Insertion of your target gene will result in increasing in the size of the PCR product. This increase corresponds to the size of your gene of interest.
Reagents and solutions
· 50x TAE (per liter) 242 g Tris base
· 57.1 ml glacial acetic acid
· 100 ml 0.5 M EDTA pH 8.0
· 6x loading buffer: 0.25% bromophenol blue
· 0.25% xylene cyanol FF
· 40% (w/v) sucrose in water
· Store at 4oC
Screening the Transposition of Your Gene of Interest into the Viral Genome
1. Prepare selective agar plates as in the electroporation and transposition procedure.
2. Label the appropriate number of 0.5 ml microcentrifuges tubes. Place on ice.
3. To each tube add:5 µl of 10X PCR buffer1 µl of 10 mM dNTP mix1.5 µl of 50 mM MgCl22.5 µl of primer mix (1.25 each 10 µM stock)0.5 µl Taq Polymerase (2.5 U)Add DDW to a final volume of 50 µl
4. Mix the contents of the tube by gently tapping the tube.
5. Pick a colony from the plate into the reaction mix.
6. Perform PCR as follows:a) 93oC for 3 minb) 94oC for 45 secc) 55oC for 45 secd) 72oc for 5 minperform steps b-d x40 cycles.
7. Prepare 0.7% agarose gel in 1xTAE containing 0.5 µg/ml ethidium bromide
8. Load 24µl of each sample on gel (prepare the sample as follows: 20 µl of PCR reaction + 4 µl of 6x loading buffer). Run gel at 100 mA.
9. The expected results from the PCR are as follows: Bacmid alone ~300 bp Bacmid transposed with pFastBac1 ~2300 bp Insertion of your target gene will result in increasing in the size of the PCR product. This increase corresponds to the size of your gene of interest.
Reagents and solutions
· 50x TAE (per liter) 242 g Tris base
· 57.1 ml glacial acetic acid
· 100 ml 0.5 M EDTA pH 8.0
· 6x loading buffer: 0.25% bromophenol blue
· 0.25% xylene cyanol FF
· 40% (w/v) sucrose in water
· Store at 4oC
