Team:Hawaii/PCR Amplification of pRL1383a

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Revision as of 03:12, 15 July 2008

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PCR Amplification of pRL1383a

Several regions of pRL1383a will be amplified with BioBrick based primers. These components will be used later in the construction of a pRL1383a BioBrick based vector. These parts include the aadA region of the omega interposon, the origin of vegetative replication (oriV), the mobilization proteins, and the replication proteins.

Methods

Materials Per Reaction Added in order:

3.5uL nanopure water
1uL 10uM forward/reverse primers
1uL pRL1383a as template (1:10 dilution of large-scale plasmid prep).
5uL Taq polymerase: Accusure(hot start, 68°C) and Red Taq

Note: the taq and water can be combined and aliquoted together as long as reaction is kept at 4°C.

cold blocks
pcr reaction tubes, with tops
pipette and tips

**PCR Running Conditions**
Duration of Time	T° 5 cycles	T° 30 cycles	Purpose
10 minutes	95°C	95°C	heat activate taq
30 seconds	95°C	95°C	denaturation
30 seconds	48.1°C, 53.4°C, 57.9°C	59°C, 60.6°C, 61.9°C	annealing
5 minutes	68°C	68°C	extension (1.5 minutes per kb, go with longest)
10 minutes	68°C	68°C	finishing the extension
infinity	4°C	4°C	(until you remove product)

Materials for the gel

1% agarose gel
10ug/mL Ethidium Bromide
running apparatus
gel running conditions: 95V for ~1 hour

Results

PCR Gel 1 (left) and PCR Gel 2 (right).

**PCR Gel 1**
Lane	Contents	Description	Predicted annealingT°C,5 cycles	Predicted annealing T°C,30 cycles
1	Tri-dye 10kb ladder	ok
2	omega (aadA)#1	(+), ~1kb	54.4,50.4	60.7,69.5
3	oriV #1	(+), ~0.5kb	51.3, 53	60.9, 71.2
4	mob #1	smear	53.5, 48	62, 69.8
5	rep #1	smear, low kb	53.9, 57.2	59.1, 73.5
6	(-)control	all clear
7	(+) control	bright band, ~0.5kb
8	omega #2	(+), ~1kb	54.4,50.4	60.7,69.5
9	oriV #2	(+) ~0.5kb	51.3, 53	60.9, 71.2
10	mob #2	nothing, slight smear	53.5, 48	62, 69.8

**PCR Gel 2**
Lane	Contents	Description	Predicted annealing T°C,5 cycles	Predicted annealing T°C,30 cycles
1	Tri-dye 10kb ladder	no ladder
2	rep#2	light smear	53.9, 57.2	59.1, 73.5
3	(-)control	all clear
4	(+)control	bright band ?0.5kb
5	omega #3	bright band, ?1kb	54.4,50.4	60.7,69.5
6	oriV #3	bright band, 0.5kb?	51.3, 53	60.9, 71.2
7	mob	light band, some smear, 3kb?	53.5, 48	62, 69.8
8	rep #3	band in low kb	53.9, 57.2	59.1, 73.5
9	(-) control	all clear
10	(+) control	bright band, 0.5kb?

Discussion

The gels are very warped. I did not let them dry long enough before I poured the running buffer,so some products did not run correctly therefore this experiment will be repeated on Monday when the necessary reagents are delivered.
I mixed up the temperature gradient!!! I need to be more careful about labeling my reactions.
The rep protein did not come out at all and the mob region only came out at one temperature, so these must be PCRed again to determine the correct annealing temperature.

Follow-up Experiments

7/2/08

1. A second attempt was made to PCR the rep region. An annealing temperature of 53.4° was used for the first 5 cycles and annealing temperature 60.6 was used for the subsequent 30 cycles.
2. Results: There are 2 faint bands in the low kb region. This is probably primers.

Lane 1 contains the ladder, lanes 3 through 6 contain the products of a plasmid prep, lane 7 contains the PCR of the rep region, while lanes 8 and 9 are the positive and negative control, respectively. Lane 10 contains one of NW's PCR reactions.

1. Discussion: The rep region did not amplify under these conditions. There were several suggestions made by the professors that I will try this weekend.

7/4/08

Materials:

- 5uL Red Taq (per reaction)
- 3.5uL nanopure water (per reaction)
- 1uL 10uM f/r rep region primers (per reaction)
- 1uL of either 1:100, 1:500, 1:1000 dilution pRL1383a as template (1:10 dilution of large-scale plasmid prep)
- 5uL accusure (for one reaction to test the dilution with this enzyme)

PCR Gel of rep region amplified with red taq.

**PCR Running Conditions**
Duration of Time	T° 5 cycles	T° 30 cycles	Purpose
10 minutes	95°C	95°C	heat activate taq
30 seconds	95°C	95°C	denaturation
30 seconds	54	59	annealing
5 minutes	70°C (red taq),68°C (accusure)	68°C (red taq),68°C (accusure)	extension (1.5 minutes per kb, go with longest)
10 minutes	70°C (red taq),68°C (accusure)	68°C (red taq),68°C (accusure)	finishing the extension
infinity	4°C	4°C	(until you remove product)

Results

**PCR Gel 1**
Lane	Contents	Description
1	Tri-dye 10kb ladder	faint, not well defined
2	rep 1:100 dilution	3.3kb, brighter than ladder, very thick band, smear (too much DNA?)
3	rep 1:500 dilution	3.3kb, brighter than ladder, very thick band, smear (too much DNA?)
4	rep 1:1000 dilution	3.3kb, brighter than ladder, very thick band, smear (too much DNA?)
5	(-)control	very faint band in low kb region (maybe contamination from other well
6	(+) control	faint band in 0.5 kb region
7	1:500 dilution rep region amplified by accusure	0.5kb band and band in low kb region, faint

7/7/08

rep and mob regions did not amplify with accusure, so they were amplified using red/green taq. Same conditions from 7/4 were used.

results

PCR amplification of mob and rep regions.

Discussion

rep region appears to be amplified by red taq efficiently. Need to use much less DNA. Find out what dilution to use. Accusure + some reaction stabilizing reagent might be used to get this reaction going, but as of right now, this region is not being amplified by this enzyme. This temperature seems to have worked well also.

Insanity is doing the same thing over and over again and expecting different results. - Albert Einstein

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

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 !width="150"|T&deg; 5 cycles
 !width="150"|T&deg; 30 cycles
-!width="150"|Purpose
+!width="400"|Purpose
 |-
 |10 minutes