Wiki/api.php

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test/1
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{{:Team:SUSTC-Shenzhen/templates/page-header|
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      title=Notebook|
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      subtitle=Elements of the endeavor.}}
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{{SUSTC-Shenzhen/main-content-begin}}
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{{:Team:SUSTC-Shenzhen/templates/notebook-main|
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      name=5*UAS plasmids Fill-in|
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      date=2014/8/6|
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      goal=To eliminate the EcoRI site on the 5*UAS plasmid}}
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==Materials==
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* dNTPs
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* Enzymes&Buffer:
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* EcoRI, Cutsmart Buffer
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* T4 DNA polymerase, T4 DNA polymerase Buffer
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* T4 DNA ligase, T4 DNA ligase buffer
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* Kit:
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** TIANgel Purification Kit
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==Methods==
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===Restriction digestion for 5*UAS plasmid with EcoRI===
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{| class="table"
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|-
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! Total volume !! Buffer !! DNA !! EcoRI !! ddH2O
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|-
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| 20μl || 2μl || 8μl || 1μl || 9μl
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|}
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Start time: 9:41am
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End time: 2:00pm
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===Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer===
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Loading system：
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{| class="table"
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|-
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! Total volume/well !! DNA !! Dye !! TAE
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|-
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| 24μl || 5μl || 4μl || 15μl
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|}
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Running conditions：110V ， 30mim
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DNA extraction:
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  Performed as the protocol provided by TIANgel Purification Kit, except that we eluted the DNA sample twice at the last step (as we always done before).
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Concentration of the purified DNA fragments:
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  Poly A: 24.8ng/ul
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  G-PB5: 42.2ng/ul
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===Do 5’-end fill-in with T4 DNA polymerase===
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'''Protocol'''
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# DNA should be dissolved in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 100 µM each dNTP.
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# Add 1 unit T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C.
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# Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75°C for 20 minutes .
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#: CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times will result in recessed ends due to the 3´→ 5´ exonuclease activity of the enzyme.
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'''Reaction system:'''
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{| class="table"
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!
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! Poly A
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! G-PB5
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|-
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| DNA
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| 17.8ul(441.44ng)
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| 17.8ul(751.16ng)
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|-
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| 10X
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  T4 DNA Ligase Reaction Buffer
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| 2ul
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| 2ul
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|-
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| dNTP
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| 0.2ul
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| 0.2ul
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|-
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| T4
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  polymerase
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  (3U/ul)
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| 0.2ul
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  (Theoretical
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  value: 0.1471ul)
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| 0.3ul
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  (Theoretical
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  value: 0.2503ul)
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|-
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| Total
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  volume
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| 20.3ul
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| 20.2ul
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|}
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'''EDTA'''
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We prepared 100mM EDTA stock solution.
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To stop the reaction, we added 1ul EDTA (100mM) to 19ul reaction liquid. Heat 75°C, 20min
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'''Blunt end ligation with T4 DNA ligase'''
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# Till now, the concentration of DNA
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#: Poly A reaction system：21.0ng/ul
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#: G-PB5 reaction system：35.3ng/ul
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# ligation system：
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#: {| class="table"
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!
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! DNA
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! 10X
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  buffer
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! T4
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  ligase
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! ddH2O
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|-
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| Theoretical
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  volume
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| 37.5ng
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| 2ul
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| 1ul
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| Add
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  to 20ul
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|-
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| Poly
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  A(1,2)
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| 2ul
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| 2ul
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| 1ul
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| 15ul
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|-
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| G-PB5(1,2)
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| 1ul
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| 2ul
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| 1ul
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| 16ul
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|}
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# Protocol:
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## Incubate 16°C, overnight (16h)
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## Heat inactivation, 65°C, 10min
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## Chill on ice
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# Remove EDTA:
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#: we add 1ul pfu (with MgSO4) buffer to each ligation system (totally, 4*tubes: polyA1, polyA2, G1, G2), in order to neutralize the inhibit effect of EDTA.
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# Restriction digestion using EcoRI to re-linearize those sticky end ligations, thus selecting out those successful ones.
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## Till now, the concentration of DNA
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##: Poly A reaction system：37.5ng/20ul=1.8ng/ul
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##: G-PB5 reaction system：37.5ng/20ul=1.8ng/ul
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## Digestion system:
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##: {| class="wikitable"
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!
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! Total
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  volume
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! EcoRI
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  (20U/ul)
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! DNA
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! 10X
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  NEBuffer
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! ddH2O
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|-
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| Theoretical volume
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| 10ul
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| 1
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  unit
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| X
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  ul
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  (0.1ug)
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| 1ul
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| Add
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  to 10ul
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|-
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| Poly A1 &
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  G-PB51
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| 22.1ul
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| 0.1ul
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  (2
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  unit, 显然酶过量了)
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| 20ul(大约37.5ng)
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| 2ul
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| -
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|}
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Also, design a non-digestion system as control
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Poly A2 & G-PB52 didn’t undergo restriction enzyme digestion.
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6. Transformation
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Performed as the usual protocol
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5ul ligation reaction to 50ul competent cell.
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Incubate: 2014.8.7 9:30pm~2014.8.8 2:30pm (17h)
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Results:
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1) From the picture, we can see that, there were no colonies growing on the plate that were grown with plasmid-digested bacteria（Poly A1, G1）. While for those bacteria which were transformed with undigested plasmid (the control group: Poly A2, G2), several colonies have already appeared on the agar plate.
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2) This meant the successfully transformed colonies were all false positive, of which the plasmids were all formed by sticky-end self-ligation, rather than blunt ends which should be generated via 5’ end Fill-in.
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3) Finally, in a word, our 5*UAS plasmids Fill-in has failed.
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Points that can be improved in the next time：
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1. Do not undergo Agrose gel DNA extraction after the-first-time enzyme digestion, since the buffer for digestion is also suitable to T4 polymerase. Thus, a relatively high concentration of DNA can be assured.
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2. After the polyreaction step of Fill-in, recover DNA by agarose gel DNA extraction, to remove EDTA, buffer etc. most of which might affect the following experiment.
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3. Design a concentration gradient when performing the T4 ligation step (30ng, 100ng, 200ng DNA). Make sure that there is always a DNA concentration involved in the optimum range for ligation.
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{{SUSTC-Shenzhen/main-content-end}}
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{{SUSTC-Shenzhen/wiki-footer}}
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Latest revision as of 09:21, 17 October 2014

test/1

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