Team:Hawaii/PCR Amplification of pRL1383a

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Revision as of 00:35, 4 July 2008

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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.

Materials Per Reaction Added in order:

3.5uL nanopure water
1uL 10uM forward/reverse primers
1uL pRL1383a as template (1:10 dilution of [Team:Hawaii/Header: Large-Scale Plasmid Prep from E. coli| 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.

**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

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?

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 ]

@@ Line 66: / Line 66: @@
 * <br>
+[[Image:PCR_prl1383a.JPG|right|thumb|300px|PCR Gel 1 (left) and PCR Gel 2 (right).]]
 {| border="1"
 |+ '''PCR Gel 1'''
-!width="150"|Lane
+!width="100"|Lane
-!width="150"|Contents
+!width="100"|Contents
-!width="150"|Description
+!width="100"|Description
-!width="150"|Predicted annealingT&deg;C,5 cycles
+!width="100"|Predicted annealingT&deg;C,5 cycles
-!width="150"|Predicted annealing T&deg;C,30 cycles
+!width="100"|Predicted annealing T&deg;C,30 cycles
 |-
 |1
@@ Line 136: / Line 136: @@
 |}
-[[Image:PCR_prl1383a.JPG|right|thumb|300px|PCR Gel 1 (left) and PCR Gel 2 (right).]]
 {| border="1"
 |+ '''PCR Gel 2'''
-!width="150"|Lane
+!width="100"|Lane
-!width="150"|Contents
+!width="100"|Contents
-!width="150"|Description
+!width="100"|Description
-!width="150"|Predicted annealing T&deg;C,5 cycles
+!width="100"|Predicted annealing T&deg;C,5 cycles
-!width="150"|Predicted annealing T&deg;C,30 cycles
+!width="100"|Predicted annealing T&deg;C,30 cycles
 |-
 |1