1. How to bioengineer a novel bio-photovoltaic system?
Obtain a sequence by PCR, then clone in suitable plasmid
TOPO allows directional cloning of PCR products!
Need correct ratio of amplicon to vector!

2. How to bioengineer a novel bio-photovoltaic system?
Need correct ratio of amplicon to vector!
Use the stock Salt Solution as supplied

3. How to bioengineer a novel bio-photovoltaic system?
Mix reaction gently and incubate for 5 minutes at room temperature (22–23˚C).
Note: For most applications, 5 minutes yields plenty of colonies. Depending on your needs, you can vary the length of the TOPO cloning reaction from 30 seconds to 30 minutes. For large PCR products (> 1 kb) increasing the reaction time may yield more colonies. You may store the reaction at –20˚C

4. For each transformation, you will need one vial of competent cells and two selective plates.
1. Equilibrate a water bath to 42˚ C
2. Warm the SOC medium & LB +100 μg/mL spectinomycin to 37˚C
3. Thaw on ice 1 vial of TOP10 cells for each transformation.

5. 1. Add 2 μL of the TOPO(R) cloning to a vial of One Shot(R) Chemically Competent E. coli and mix gently. Do not mix
by pipetting up and down.
2. Incubate on ice for 5 to 30 minutes.
Note: Longer incubations on ice seem to have a minimal effect on transformation efficiency.

6. 1. Add 2 μL of the TOPO(R) cloning to a vial of One Shot(R) Chemically Competent E. coli and mix gently. Do not mix
by pipetting up and down.
2. Incubate on ice for 5 to 30 minutes.
Note: Longer incubations on ice seem to have a minimal effect on transformation efficiency.
3. Heat-shock the cells for 30 seconds at 42˚C without shaking.

7. 1. Add 2 μL of the TOPO(R) cloning to a vial of One Shot(R) Chemically Competent E. coli and mix gently. Do not mix
by pipetting up and down.
2. Incubate on ice for 5 to 30 minutes.
Note: Longer incubations on ice seem to have a minimal effect on transformation efficiency.
3. Heat-shock the cells for 30 seconds at 42˚C without shaking.
4. Immediately transfer the tubes to ice.
5. Add 250 μL of room temperature S.O.C. Medium.

8. 1. Add 2 μL of the TOPO(R) cloning to a vial of One Shot(R) Chemically Competent E. coli and mix gently. 2. Incubate on ice for 5 to 30 minutes.
3. Heat-shock the cells for 30 seconds at 42˚C without shaking.
4. Immediately transfer the tubes to ice.
5. Add 250 μL of room temperature S.O.C. Medium.
6. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37˚C for > 30 minutes.

9. Transfer cells & grow >30’ @ 37˚ C with shaking
Plate 10 & 100 µl on LB mg/l spectinomycin & grow 37˚C

10. Cloning Orthologs
Design primers to obtain entire CDS + minimal flanking sequence from suitable source!
Design nested primers that start at ATG and end at stop codon and add CACC at 5’ end

11. Cloning via TOPO?
Obtain a sequence by PCR, then clone in suitable plasmid
Topoisomerases : enzymes that untie knots in DNA

12. Cloning via TOPO?
Obtain a sequence by PCR, then clone in suitable plasmid
Topoisomerases : enzymes that untie knots in DNA
Type I nick backbone & unwind once as strand rotates
Relieve (or increase) supercoiling

13. Cloning via TOPO?
Obtain a sequence by PCR, then clone in suitable plasmid
Topoisomerases : enzymes that untie knots in DNA
Type I nick backbone & unwind once as strand rotates
Type II cut both strands: relieve two supercoils/rxn

14. Cloning via TOPO?
Obtain a sequence by PCR, then clone in suitable plasmid
Topoisomerases : enzymes that untie knots in DNA
Type I knick backbone & unwind once as strand rotates
Type II cut both strands: relieve two supercoils/rxn
Separate plasmids that became interwoven during DNA replication

15. Cloning via TOPO?
Obtain a sequence by PCR, then clone in suitable plasmid
Topoisomerases : enzymes that untie knots in DNA
Type I nick backbone & unwind once as strand rotates
Type II cut both strands: relieve two supercoils/rxn
DNA gyrase plays important role in DNA replication by relieving supercoils created by unwinding DNA

16. How to bioengineer a novel bio-photovoltaic system?
Obtain a sequence by PCR, then clone in suitable plasmid
Vaccinia virus topoisomerase I binds DNA at specific sites and cleaves the backbone after 5′-CCCTT in one strand

17. How to bioengineer a novel bio-photovoltaic system?
Obtain a sequence by PCR, then clone in suitable plasmid
Vaccinia virus topoisomerase I binds DNA at specific sites and cleaves the backbone after 5′-CCCTT in one strand
TOPO allows directional cloning of PCR products!

18. How to bioengineer a novel bio-photovoltaic system?
Obtain a sequence by PCR, then clone in suitable plasmid
Vaccinia virus topoisomerase I binds DNA at specific sites and cleaves the backbone after 5′-CCCTT in one strand
The energy is conserved by bonding the cleaved strand to Tyr-274 of topo I.
This bond can be
reversed, releasing
topoisomerase. TOPO
cloning uses this
reaction to clone
PCR products.

19. How to bioengineer a novel bio-photovoltaic system?
This bond can be reversed, releasing topoisomerase. TOPO cloning uses this reaction to clone PCR products.
DNA is joined by adding a 3′ overhang to the new DNA
complementary to the 5′ end of the TOPO-charged DNA.

20. How to bioengineer a novel bio-photovoltaic system?
DNA is joined by adding a 3′ overhang to the new DNA
complementary to the 5′ end of the TOPO-charged DNA.
The GTGG overhang in the cloning vector invades the
5′ end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation.

21. How to bioengineer a novel bio-photovoltaic system?
DNA is joined by adding a 3′ overhang to the new DNA
complementary to the 5′ end of the TOPO-charged DNA.
The GTGG overhang in the cloning vector invades the
5′ end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation.
DNA ligase then links the
fragments

22. How to bioengineer a novel bio-photovoltaic system?
Obtain a sequence by PCR, then clone in suitable plasmid
TOPO allows directional cloning of PCR products!
Transform product into E.coli
Identify clones by PCR
Confirm by sequencing

23. Verifying insert
Pick 10 colonies into Terrific broth mg/l spectinomycin

24. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spectinomycin
Grow 37 ˚ C

25. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g

26. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE

27. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE

28. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE
Resuspend in 90 µl GTE

29. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.

30. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.
Add 200 µl 1% SDS in 200 mM NaOH & rock back & forth until tube clears.

31. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.
Add 200 µl 1% SDS in 200 mM NaOH & rock back & forth until tube clears.
Add 150 µl of 10 M ammonium acetate and place on ice.

32. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.
Add 200 µl 1% SDS in 200 mM NaOH & rock back & forth until tube clears.
Add 150 µl of 10 M ammonium acetate and place on ice.
Wait 10’, then spin 14 kg

33. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.
Add 200 µl 1% SDS in 200 mM NaOH & rock back & forth until tube clears.
Add 150 µl of 10 M ammonium acetate and place on ice.
Wait 10’, then spin 14 kg
Transfer supernatant to new tube, add 500 µl Isoprop

34. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.
Add 200 µl 1% SDS in 200 mM NaOH & rock back & forth until tube clears.
Add 150 µl of 10 M ammonium acetate and place on ice.
Wait 10’, then spin 14 kg
Transfer supernatant to new tube, add 500 µl Isoprop

35. Verifying insert
Pick 10 white colonies into Terrific broth mg/l spect
Grow 37 ˚ C
Harvest x g
Resuspend in 1 ml GTE
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.
Add 200 µl 1% SDS in 200 mM NaOH & rock back & forth until tube clears.
Add 150 µl of 10 M ammonium acetate and place on ice.
Wait 10’, then spin 14 kg
Transfer supernatant to new tube, add 500 µl Isoprop
Dump supernatant, wash pellet with 200 µl 70% EtOH

36. Verifying insert
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.
Add 200 µl 1% SDS in 200 mM NaOH & rock back & forth until tube clears.
Add 150 µl of 10 M ammonium acetate and place on ice.
Wait 10’, then spin 14 kg
Transfer supernatant to new tube, add 500 µl Isoprop
Dump supernatant, wash pellet with 200 µl 70% EtOH
Remove as much supernatant as possible, then air-dry

37. Verifying insert
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.
Add 200 µl 1% SDS in 200 mM NaOH & rock back & forth until tube clears.
Add 150 µl of 10 M ammonium acetate and place on ice.
Wait 10’, then spin 14 kg
Transfer supernatant to new tube, add 500 µl Isoprop
Dump supernatant, wash pellet with 200 µl 70% EtOH
Remove as much supernatant as possible, then air-dry
Resuspend in 100 µl TE + 20 µg/ml RNAse A

38. Verifying insert
Resuspend in 90 µl GTE
Add 10µl 2 mg/ml lysozyme in GTE & leave 5 minutes.
Add 200 µl 1% SDS in 200 mM NaOH & rock back & forth until tube clears.
Add 150 µl of 10 M ammonium acetate and place on ice.
Wait 10’, then spin 14 kg
Transfer supernatant to new tube, add 500 µl Isoprop
Dump supernatant, wash pellet with 200 µl 70% EtOH
Remove as much supernatant as possible, then air-dry
Resuspend in 100 µl TE + 20 µg/ml RNAse A
Test by restriction and PCR as per your plans