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-       name=5*UAS plasmids Fill-in|
+</html>
-       date=2014/8/6|
-       goal=To eliminate the EcoRI site on the 5*UAS plasmid}}
-==Materials==
-* dNTPs
-* Enzymes&Buffer:
-* EcoRI, Cutsmart Buffer
-* T4 DNA polymerase, T4 DNA polymerase Buffer
-* T4 DNA ligase, T4 DNA ligase buffer
-* Kit:
-** TIANgel Purification Kit
-==Methods==
-===Restriction digestion for 5*UAS plasmid with EcoRI===
-{| class="table"
-|-
-! Total volume !! Buffer !! DNA !! EcoRI !! ddH2O
-|-
-| 20μl || 2μl || 8μl || 1μl || 9μl
-|}
-Start time: 9:41am
-End time: 2:00pm
-===Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer===
-Loading system：
-{| class="table"
-|-
-! Total volume/well !! DNA !! Dye !! TAE
-|-
-| 24μl || 5μl || 4μl || 15μl
-|}
-Running conditions：110V ， 30mim
-DNA extraction:
-  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).
-Concentration of the purified DNA fragments:
-   Poly A: 24.8ng/ul
-   G-PB5: 42.2ng/ul
-===Do 5’-end fill-in with T4 DNA polymerase===
-'''Protocol'''
-# DNA should be dissolved in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 100 µM each dNTP.
-# Add 1 unit T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C.
-# Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75°C for 20 minutes .
-#: 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.
-'''Reaction system:'''
-{| class="table"
-!
-! Poly A
-! G-PB5
-|-
-| DNA
-| 17.8ul(441.44ng)
-| 17.8ul(751.16ng)
-|-
-| 10X
-  T4 DNA Ligase Reaction Buffer
-| 2ul
-| 2ul
-|-
-| dNTP
-| 0.2ul
-| 0.2ul
-|-
-| T4
-  polymerase
-  (3U/ul)
-| 0.2ul
-  (Theoretical
-  value: 0.1471ul)
-| 0.3ul
-  (Theoretical
-  value: 0.2503ul)
-|-
-| Total
-  volume
-| 20.3ul
-| 20.2ul
-|}
-'''EDTA'''
-We prepared 100mM EDTA stock solution.
-To stop the reaction, we added 1ul EDTA (100mM) to 19ul reaction liquid. Heat 75°C, 20min
-'''Blunt end ligation with T4 DNA ligase'''
-# Till now, the concentration of DNA
-#: Poly A reaction system：21.0ng/ul
-#: G-PB5 reaction system：35.3ng/ul
-# ligation system：
-#: {| class="table"
-!
-! DNA
-! 10X
-  buffer
-! T4
-  ligase
-! ddH2O
-|-
-| Theoretical
-  volume
-| 37.5ng
-| 2ul
-| 1ul
-| Add
-  to 20ul
-|-
-| Poly
-  A(1,2)
-| 2ul
-| 2ul
-| 1ul
-| 15ul
-|-
-| G-PB5(1,2)
-| 1ul
-| 2ul
-| 1ul
-| 16ul
-|}
-# Protocol:
-## Incubate 16°C, overnight (16h)
-## Heat inactivation, 65°C, 10min
-## Chill on ice
-# Remove EDTA:
-#: 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.
-# Restriction digestion using EcoRI to re-linearize those sticky end ligations, thus selecting out those successful ones.
-## Till now, the concentration of DNA
-##: Poly A reaction system：37.5ng/20ul=1.8ng/ul
-##: G-PB5 reaction system：37.5ng/20ul=1.8ng/ul
-## Digestion system:
-##: {| class="wikitable"
-!
-! Total
-  volume
-! EcoRI
-  (20U/ul)
-! DNA
-! 10X
-  NEBuffer
-! ddH2O
-|-
-| Theoretical volume
-| 10ul
-| 1
-  unit
-| X
-  ul
-  (0.1ug)
-| 1ul
-| Add
-  to 10ul
-|-
-| Poly A1 &
-  G-PB51
-| 22.1ul
-| 0.1ul
-  (2
-  unit, 显然酶过量了)
-| 20ul(大约37.5ng)
-| 2ul
-| -
-|}
-Also, design a non-digestion system as control
-Poly A2 & G-PB52 didn’t undergo restriction enzyme digestion.
-. Transformation
-Performed as the usual protocol
-ul ligation reaction to 50ul competent cell.
-Incubate: 2014.8.7 9:30pm~2014.8.8 2:30pm (17h)
-Results:
-) 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.
-) 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.
-) Finally, in a word, our 5*UAS plasmids Fill-in has failed.
-Points that can be improved in the next time：
-. 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.
-. 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.
-. 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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