Lab 208

Main purpose

• Colony PCR from Pseudomonas aeruginosa (PAO1) to isolate Nir.
• Colony PCR to check the presence of AMO and HAO in transformants E.coli.

Gel on yesterdays PCR Isolation of genomic DNA from PAO1

Who was in the lab

Kristian, Gosia, Henrike, Julia

Procedure

Colony PCR

Colony PCR was performed according to standard method in order to check if transformed cells which grew on medium contain desired insert in pZA21 plasmid. The colonies were chosen from agar plate to be checked. Names and numbers of colonies as well as plates names and primers are:

• HAO 1-6 in pZA21 USER, primers 28a, 28b
• AMO 1-9 in pZA21 USER, primers 29a, 29b

PCR programms based on standard PCR programm with annealing temperatures 52C for HAO and 56C for AMO and extension time 3:00 mins.

Colony PCR for Nir

In order to extract Nir from Pseudomonas we used primers which do not contain uracil and new PCR programm (touch-down PCR). Phusion polymerase was used.

Names of samples are as follows:

• 1,2 - primers 41a, 41b - Nir, part 1
• 3,4 - primers 42a, 42b - Nir, part 2
• 5,6 - primers 11a, 11b - all fragment

PCR programm (write it later when programm will be done)

Results

Gel of PCR products from yesterday

• 1 - 1kb ladder
• 2 - Nir, part 1
• 3 - Nir, part 2
• 4 - Nir, part 1
• 5 - Nir, part 2
• 6 - Nir, part 1
• 7 - Nir, part 2
• 8 - Nir, part 1
• 9 - Nir, part 2
• 10 - pZE21 without promotor
• 11 - pZE21 without promotor
• 12 - HAO
• 13 - HAO
• 14 - negative
• 15 - 1 kb ladder

Gel of PCR to extract Nir from Pseudomonas

products of todays touchdown PCR

• 1 - 1 kb ladder
• 2 - Nir part 1
• 3 - Nir part 1
• 4 - Nir part 2
• 5 - Nir part 2
• 6 - broad band ladder

break

• 7 - 1 kb ladder
• 8 - Nir whole fragment
• 9 - Nir whole fragment
• 10 - broad band ladder

Gel of todays colony PCR

Colony PCR to check for inserts in tranformants from USER reaction on the 24.07.

• 1 - 1 kb ladder
• 2 - AMO 1
• 3 - AMO 2
• 4 - AMO 3
• 5 - AMO 4
• 6 - AMO 5
• 7 - AMO 6
• 8 - AMO 7
• 9 - AMO 8
• 10 - AMO 9
• 11 - 1 kb ladder
• 12 - HAO 1
• 13 - HAO 2
• 14 - HAO 3

broken well

• 16 - HAO 4
• 17 - HAO 5
• 18 - HAO 6
• 19 - 1 kb ladder

faint bands of ~ 3000 kb can be seen in AMO colonies 2 and 3

Genomic DNA isolation

Followed the protocol from the Qiagen: DNeasy Blood & Tissue Kit to isolate genomic DNA from the first PAO1 culture we got. Followed up by splitting the sample of purified DNA and treating one of them with HindIII which have no restriction sites within the Nir genes we are interested in. Then made PCR with primer set 11 on 65C and 4:30 extension. The reactions had either pure genomic DNA or HindIII treated and had a gradient of concentrations: 1, 3, 7, 10, 12, 15uL of DNA in place of MQ.

Conclusion

PCR of Nir fragment gave a lot of unspecific products. New programm and new components for PCR are used to amplify it.

Innoculated ON cultures of AMO colony 2 and colony 3 for restriction analysis tomorrow.

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