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| - | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_HEAD}}
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| - | This is a placeholder. We should fill it in.
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| - | ==Construct Design==
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| - | ==Primer Design==
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| - | We should mention expected lengths of products here.
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| - |
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| - | ==Assembly: first attempt==
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| - | ===PCR===
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| - | In the first round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3 and GA4 constructs.
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| - | *We performed PCR using [[Team:Cambridge/Protocols/PCR#Reagents_used_in_PCR_reaction | Phusion Hot Start DNA Polymerase]] in 20 μl reaction volume.
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| - | *The time profile used in the PCR machine was the following:
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| - | {| border="1" align="center" style="text-align:center;"
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| - | |scope="col" width="50" | '''Hold'''
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| - | |95°C
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| - | |2 min
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| - | |-
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| - | |scope="col" width="50" rowspan="3" | '''Cycling'''
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| - | |scope="col" width="80" | ''Denaturing''
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| - | |scope="col" width="50" | 95°C
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| - | |scope="col" width="50" | 10 s
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| - | |-
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| - | |scope="col" width="80" | ''Annealing''
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| - | |scope="col" width="50" | 55°C
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| - | |scope="col" width="50" | 20 s
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| - | |-
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| - | |scope="col" width="80" | ''Elongation''
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| - | |scope="col" width="50" | 72°C
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| - | |scope="col" width="50" | 150 s
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| - | |}
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| - | We decided to use the 55°C annealing temperaure, although the predicted temperature for most primers is 5-10°C higher, because of a low annealing temperature of the VF2 primer.
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| - | *Primers and template DNA provided by our supervisor Paul served as a positive control, but eventually we did not detect any products on the gels.
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| - |
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| - | The pictures below present result of gel electrophoresis of PCR products.
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| - | *In most cases position of a band matches the expected length of DNA fragment. The only exception are GA1-2 and GA3-2 products. According to the position on the gel the length of these DNA fragments is 4-5kb, whereas the predicted length is 3.5kb. Our hypothesis is that we were provided [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] backbone instead of [http://partsregistry.org/Part:pSB1A3 pSB1A3]backbone.
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| - | *For GA1-1, GA2-1, GA3-1 and GA4-1 we obtained two bands: 1000bp and 400bp, with the latter resulting from non-specific priming most probably. We extracted the two bands for GA1-1, GA2-1 and GA4-1 products, labelling the 1000kb and 400bp fragments GAX-1a and GAX1-b respectively.
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| - | *The molecular weight marker that we used in all gels is HyperLadder I, which produces regularly spaced bands ranging from 200 to 10,000bp.
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| - | For the gel extraction of DNA we followed the [[Team:Cambridge/Protocols/Gel_Extraction_of_DNA | protocol]], assuming that one slice of gel is 100μl.
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| - |
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| - | ===Gibson Assembly===
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| - | We conducted Gibson Assembly of GA1, GA2, GA3 and GA4 constructs according to [Team:Cambridge/Protocols/Gibson_Assembly#Practice | the protocol]. The volumes of Master Mix and solutions of amplified DNA were the following:
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| - |
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| - | {| border="1" align="center" style="text-align:center;"
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| - | !scope="col" width="150" | GA1
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| - | !scope="col" width="150" | GA2
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| - | !scope="col" width="150" | GA3
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| - | !scope="col" width="150" | GA4
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| - | |-
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| - | |15µl Master Mix
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| - | |15µl Master Mix
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| - | |15µl Master Mix
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| - | |15µl Master Mix
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| - | |-
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| - | |2.5µl GA1-1a
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| - | |1.67µl GA2-1a
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| - | |2.5µl GA3-1
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| - | |1.67µl GA4-1a
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| - | |-
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| - | |2.5µl GA1-2
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| - | |1.67µl GA2-2
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| - | |2.5µl GA3-2
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| - | |1.67µl GA4-2
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| - | |-
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| - | |
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| - | |1.67µl GA2-3
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| - | |
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| - | |1.67µl GA4-3
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| - | |}
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| - |
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| - | ===Transformation===
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| - | We transformed competent ''E.coli'' cells according to [Team:Cambridge/Protocols/Transformation_of_E.Coli | the following protocol].
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| - | We cultured each class of transformants on four different plates.
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| - | {|
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| - | !scope="col" width="220" |
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| - | !scope="col" width="180" | GA1 and GA3
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| - | !scope="col" width="200" | GA2 and GA4
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| - | |-
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| - | |Plate 1 (10μl of cell suspension)
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| - | |LB + ampicillin
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| - | |LB + kanamycin
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| - | |-
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| - | |Plate 2 (100μl of cell suspension)
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| - | |LB + ampicillin
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| - | |LB + kanamycin
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| - | |-
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| - | |Plate 3 (10μl of cell suspension)
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| - | |LB + ampicillin + arabinose
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| - | |LB + kanamycin + arabinose
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| - | |-
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| - | |Plate 4 (100μl of cell suspension)
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| - | |LB + ampicillin + arabinose
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| - | |LB + kanamycin + arabinose
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| - | |}
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| - |
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| - | ===Results===
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| - | ===Diagnostics===
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| - | ==Assembly: second attempt==
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| - | ===PCR===
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| - | In the second round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3, GA4 as well as GA5 and GA6 constructs.
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| - | We ran PCR products on a 1% agarose gel to separate amplified products from template DNA and primers, as well as to check how efficient and specific the amplification process was.
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| - | *All components of GA5 and GA6 constructs produced clear fairly thick bands with positions matching expected lengths. No non-specific bands on GA5-1 (Reflectin A2) and GA6-1 (Reflectin 1B) lanes were observed.
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| - | *The molecular weight marker that we used in all gels is HyperLadder I, which produces regularly spaced bands ranging from 200 to 10,000bp.
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| - |
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| - | ===Gibson Assembly===
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| - | ===Transformation===
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| - | ===Results===
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| - | ==What next?==
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| - |
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| - | {{Template:Team:Cambridge/CAM_2011_TEMPLATE_FOOT}}
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