Matt Gethers/CRI, Thailand/Labwork/PCRs/Amplification of HmgR ORF

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Amplification of HmgR ORF

Rxn Mix

Reagent Volume/Amount
Paeru Genomic DNA Template 0.5 μl from So Pa Pan stock
Pfu 0.5 μl
Primer BT2736 4 μl 1 μM Stock
Primer BT2737 4 μl 1 μM Stock
dNTPs 1 μl 10 mM Stock
DMSO 2.5 μl
Buffer S 5 μl
dH20 32.5 μl
Total 50 μl
Reagent Volume/Amount
Paeru Genomic DNA Template 0.5 μl from So Pa Pan stock
Taq 0.5 μl
Primer BT2736 4 μl 1 μM Stock
Primer BT2737 4 μl 1 μM Stock
dNTPs 1 μl 10 mM Stock
DMSO 2.5 μl
Buffer 5 μl
Mg2+ 2.5 μl
dH20 30 μl
Total 50 μl

Rxn Conditions

Annealing Temperature: 55.0oC

Extension Time: 2 minutes, 1 minute (2 minutes/Kb for Pfu, 1 minute/Kb Taq)

Cycle (Pfu)

Step Temperature Duration Notes
Initial Denaturation 95oC 3 minutes
Repeat Cyclic Steps 35x
Cyclic Denaturation 94oC 40 Seconds
Cyclic Annealing 55oC 40 Seconds Unsure of best temp
Cyclic Extension 72oC 2 minutes
Repeat Cyclic Steps 35x
Final Extension 72oC 10 minutes

Cycle (Taq)

Step Temperature Duration Notes
Initial Denaturation 95oC 2 minutes
Repeat Cyclic Steps 35x
Cyclic Denaturation 94oC 30 Seconds
Cyclic Annealing 55oC 30 Seconds Unsure of best temp
Cyclic Extension 72oC 1 minute
Repeat Cyclic Steps 35x
Final Extension 72oC 10 minutes

Run Notes

6.17.08

Ran the protocol as written except the extension time was 3 minutes rather than 2 - I'm running the 2736/2737 PCR alongside the 1033/2724 PCR which requires the 3 minute extension.

6.18.08

The last attempt to amplify the HmgR ORF failed and I'm guessing it's due to a suboptimal annealing temp. I ran a gradient PCR this morning for the annealing temp between 50oC and 65oC. While I had originally intended to have 10 μl samples, I some how turned up short with the master mix - I checked the math and it was correct, so I must have pipetted something improperly. In any case, the end result was 8 6.25 μl reactions of the 2736/2737 primer mix and 1 10 μl reaction of the 1033/2724 primer mix (positive control). I placed the positive control at the 52.5oC well - this annealing temp is ~3 degrees lower than what I've been using, but I hope it will be ok. The other thing I noticed is that the thermocycler only allows you to set the reaction volume at a minimum of 15 μl, so that could cause some problems. Depending on the results, this gradient PCR may be worth repeating.

6.23.08 Part I

This was screening colony PCRs for the HmgR ORF among transformants (had the pET-11a plasmid). I used 7.5 μl 2x PCR mastermix, 1.2 μl of each primer (BT2736 and BT2737), and 5.1 μl colony suspension (1 colony in 50 μl H20). For the positive control, I used 0.5 μl of the genomic DNA template for Paeru and added 4.6 μl H20 to bring it up to volume. I screened six colonies. The annealing temp was 55oC and the extension time was 2 minutes.

6.23.08 Part II

Because the positive control lane looked odd ( see notes on gel here), I reran the colony screening using Pfu in stead. I used the same Pfu reaction mix described above except that I used 15 μl reactions. I screened 5 colonies and reserved one reaction for the positive control using Pfu and genomic DNA template, and one reaction for the supermix using genomic DNA template. 55oC annealing temp, 2 minute extension time.

6.24.08

I've tentatively found one colony that looks to have the HmgR ORF in pET-11a, but to be safe, I should look for at least one more (anything could be wrong with my one colony). I'm doing a second round of screening to see if I find another candidate. In addition, I'm repeating the PCR on colony 5 from the first round of screening to be sure that it does in fact have the correct product. I'm also including a positive control using genomic DNA template. Pfu is expensive, so I'm going back to the supermix, but I've taken a fresh aliquot - hopefully that will resolve the problem with the positive control (and reveal any more candidates). The reaction mix was 7.5 μl supermix, 1.2 μl each primer (BT2736 and BT2737), 4.1 μl H20, and 1 μl colony suspension (1 colony in 50 μl H20). For the positive control, I used 0.5 μl genomic DNA template and added 0.5 μl H20 to bring the reaction up to volume.

6.25.08

The supermix just isn't working for me, so I'm using a Taq PCR kit from Finnzyme instead. Made a master mix of water, magnesium, buffer, DMSO, dNTPs, and Taq, split in two, then added primers M13 for and BT2724 to one aliquot and primers BT2736 an dBT2737 to the other aliquot. Then I aliquotted 8.2 μl to each of 12 tubes, 6 for pUC18/HmgA Downstream colonies, 5 for pET-11a/HmgR, and 1 for a positive control using Genomic DNA (still using BT2736/37). I modified the protocol a bit - I made 10 μl reactions and I used 1 μl of template for each 10 μl reaction (except for the positive control in which I used 0.5 μl temp and 0.5μl water). So rather than using 5.9 μl water per reaction, I used 5.1. I'm running with an annealing temp of 55 degrees and an extension time of 1:15 minutes (taq requires shorter time than Pfu and I'm running this PCR along side the HmgA Downstream fragment PCR, hence the slightly longer time).

6.25.08

Using the Taq PCR kit from Finnzyme again, Made a master mix of water, magnesium, buffer, DMSO, dNTPs, primers BT2736 for and BT2737, and Taq, split in two, then added 1 μl colony 5 suspension (from way back when - the first and only thing to show product) to one aliquot and 0.5 μl genomic DNA and 0.5 μl H20 to the other. I made 10 μl reactions. I'm running with an annealing temp of 55 degrees and an extension time of 50 seconds.

6.28.08

I ran a gradient PCR (between 50 and 65 degrees) looking for the optimum annealing temperature of BT2736/37. I used the Taq settings, but I allowed 1 minute for extension rather than 50 seconds. 10 μl reactions using the reaction mix for taq.

6.29.08

Given that all annealing temps yielded product for the 6.28.08 PCR, I assume that the 1 minute extension time is the critical parameter. I'm starting 7 10 μl colony PCRs from the pIs001.3, transformation from Friday along with one positive control (genomic DNA), all using BT2736 and BT2737. I'm using a 55 degree annealing temp and a 1 minute extension time at 72 degrees. All the other conditions are as written for Taq in the protocol above.

7.25.08

Did colony PCR on 7 colonies selected from 7.24.08 transformation of pIs001.L3 into DH5α and one Positive control using PAO1 prep. Made a master mix, aliquotted 9 μl to each of 8 tubes, and then added 1 μl colony suspension to 7 tubes, and 0.5 μl genomic DNA to the eighth as the positive control. Used 55 degree annealing temp and 1 minute extension time. Gel Results Here.

8.3.08

Did colony PCRs on 8.2.08 transformants. Looking for HmgR. Made a master mix and aliquotted 9 μl to 8 tubes. Four were for pIs001.L4 and the remaining four were for pIs001.L5. Added 1 μl of colony suspension (1 colony in 50 μl water). Ran PCR conditions as stated in protocol for Taq. Gel results here.

8.4.08

Did colony PCRs on 8.2.08 transformants again. Looking for HmgR. Made a master mix and aliquotted 9 μl to 15 tubes (meant for 16, but fell short). Eight were for pIs001.L4, seven were for pIs001.L5, and one was for the positive control (genomic DNA). Added 1 μl of colony suspension (1 colony in 25 μl water) and 0.5 μl of genomic DNA. Ran PCR conditions as stated in protocol for Taq. Gel results here.

8.6.08

Did colony PCRs on 8.5.08 transformants . Looking for HmgR. Made a master mix and aliquotted 9 μl to 15 tubes (meant for 16, but fell short). Eight were for pIs001.L6, seven were for pIs001.L7, and one was for the positive control (genomic DNA). Added 1 μl of colony suspension (1 colony in 25 μl water) and 0.5 μl of genomic DNA. Ran PCR conditions as stated in protocol for Taq. Gel results here.

8.7.08

Did colony PCRs on 8.6.08 transformants . Looking for HmgR. Made a master mix and aliquotted 9 μl to 9 tubes. Eight were for pIs001.L8 and one was for the positive control (genomic DNA). Added 1 μl of colony suspension (1 colony in 25 μl water) and 0.5 μl of genomic DNA. Ran PCR conditions as stated in protocol for Taq. Gel results here.

8.11.08

Did colony PCRs on more 8.6.08 transformants. Looking for HmgR. Made a master mix and aliquotted 9 μl to 7 tubes. Two were for pIs001.P7 and P8, two were for colonies '4' and '5' from the original pIs001.L8 transformant colony suspensions while two more were for colonies from the 8.6.08 transformation that were freshly suspended in water. One was for the genomic template positive control. Added 1 μl of colony suspension (1 colony in 25 μl water), prep, and genomic DNA. Ran PCR conditions as stated in protocol for Taq. Gel results here.

8.12.08

Did colony PCRs on more 8.11.08 transformants. Looking for HmgR. Made a master mix and aliquotted 9 μl to 9 tubes. 8 were colonies from the transformation while 1 was the genomic template positive control. Added 1 μl of colony suspension (1 colony in 50 μl water) and 0.5 μl genomic DNA. Ran PCR conditions as stated in protocol for Taq. Gel results here.

References

Taq PCR II