Team:Hawaii/PCR Amplification of pRL1383a

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Contents

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 [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.

  • 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

  • What was learned and how to do future experiments differently.
Insanity is doing the same thing over and over again and expecting different results. - Albert Einstein


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