Team:Hawaii/PCR Amplification of pRL1383a

From 2008.igem.org

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(PCR on 7/18)
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<strong>PCR Clean-up</strong>
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MinElute PCR Purification Kit Protocol from Qiagen
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using a microcentrifuge
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This protocol is designed to purify double-stranded DNA fragments from PCR reactions
 +
resulting in high end-concentrations of DNA (see page 12). Fragments ranging from
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70 bp to 4 kb are purified from primers, nucleotides, polymerases, and salts using
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MinElute spin columns in a microcentrifuge.
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# Mix PB 5:1 PCR reaction.
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# Place a MinElute column in a provided 2 ml collection tube.
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# Apply ALL of sample to the MinElute column; centrifuge (13,000rpm)1 min.
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# Discard flow-through. Place the MinElute column back into the same tube.
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# Add 750 μl Buffer PE to the MinElute column; centrifuge (13,000rpm) 1 min.
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#Discard flow-through and place the MinElute column back in the same tube.
 +
Centrifuge the column for an additional 1 min at maximum speed.
 +
#Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
 +
#Add 10 μl 1x TE to the center of the membrane, let the column stand for 1 min; centrifuge 1 min.
=== Discussion ===
=== Discussion ===
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<blockquote>''Insanity is doing the same thing over and over again and expecting different results.'' - Albert Einstein</blockquote>
<blockquote>''Insanity is doing the same thing over and over again and expecting different results.'' - Albert Einstein</blockquote>
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<strong>7/18/08</strong>
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:*PCR amplification of oriV, omega interposon, aadA gene, & rep/mob all using green taq.
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:* PCR conditions: 2min@94&deg;C, 30sec@95&deg;C, 30sec@50.4&deg;C, 5min30sec@72&deg;C(1 minute per kilobase), infinity@4&deg;C
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{| border="1"
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|+ '''PCR Products'''
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!width="100"|Name
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!width="100"|Completed PCR
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!width="100"|Cleaned?
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!width="100"|Description
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|-
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|OriV
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|no
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|no
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|origin of vegetative replication
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|-
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|rep region
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|yes
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|no
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|proteins necessary for autonomous replication
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|-
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|mob region
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|yes
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|no
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|mobilization proteins & origin of transfer
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|-
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|rep/mob region
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|no
 +
|no
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|mob and rep proteins/necessary for mobilization by Ti plasmid
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|-
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|omega interposon
 +
|no
 +
|no
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|insertional mutagenesis & Sm, Sp resistance
 +
|-
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|aadA gene
 +
|no
 +
|no
 +
|Sm, Sp resistance
 +
|}
{{Team:Hawaii/Footer}}
{{Team:Hawaii/Footer}}

Revision as of 23:04, 18 July 2008

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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 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?

PCR Clean-up

MinElute PCR Purification Kit Protocol from Qiagen using a microcentrifuge

This protocol is designed to purify double-stranded DNA fragments from PCR reactions resulting in high end-concentrations of DNA (see page 12). Fragments ranging from 70 bp to 4 kb are purified from primers, nucleotides, polymerases, and salts using MinElute spin columns in a microcentrifuge.

  1. Mix PB 5:1 PCR reaction.
  2. Place a MinElute column in a provided 2 ml collection tube.
  3. Apply ALL of sample to the MinElute column; centrifuge (13,000rpm)1 min.
  4. Discard flow-through. Place the MinElute column back into the same tube.
  5. Add 750 μl Buffer PE to the MinElute column; centrifuge (13,000rpm) 1 min.
  6. Discard flow-through and place the MinElute column back in the same tube.

Centrifuge the column for an additional 1 min at maximum speed.

  1. Place the MinElute column in a clean 1.5 ml microcentrifuge tube.
  2. Add 10 μl 1x TE to the center of the membrane, let the column stand for 1 min; centrifuge 1 min.

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 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)


PCR Gel of rep region amplified with red taq.
  • 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

  1. 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

  1. mob and rep regions were amplified by both red and green taq. Use green in the future, it is cheaper.
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

7/18/08

  • PCR amplification of oriV, omega interposon, aadA gene, & rep/mob all using green taq.
  • PCR conditions: 2min@94°C, 30sec@95°C, 30sec@50.4°C, 5min30sec@72°C(1 minute per kilobase), infinity@4°C
PCR Products
Name Completed PCR Cleaned? Description
OriV no no origin of vegetative replication
rep region yes no proteins necessary for autonomous replication
mob region yes no mobilization proteins & origin of transfer
rep/mob region no no mob and rep proteins/necessary for mobilization by Ti plasmid
omega interposon no no insertional mutagenesis & Sm, Sp resistance
aadA gene no no Sm, Sp resistance

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