5*UAS plasmids Fill-in

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

Start time: 9:41am End time: 2:00pm

Electrophoresis & Agrose gel DNA extraction to remove enzymes and competent of Buffer

Loading system：

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

1. DNA should be dissolved in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 100 µM each dNTP.
2. Add 1 unit T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C.
3. 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:

 T4 DNA Ligase Reaction Buffer

 polymerase
 (3U/ul)

 (Theoretical
 value: 0.1471ul)

 (Theoretical
 value: 0.2503ul)

 volume

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

1. Till now, the concentration of DNA

   Poly A reaction system：21.0ng/ul

   G-PB5 reaction system：35.3ng/ul

2. 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 |}

1. Protocol:
   1. Incubate 16°C, overnight (16h)
   2. Heat inactivation, 65°C, 10min
   3. Chill on ice
2. 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.

3. Restriction digestion using EcoRI to re-linearize those sticky end ligations, thus selecting out those successful ones.
   1. 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

   2. 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. 6. Transformation Performed as the usual protocol 5ul ligation reaction to 50ul competent cell. Incubate: 2014.8.7 9:30pm~2014.8.8 2:30pm (17h) Results:

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. 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. 3) Finally, in a word, our 5*UAS plasmids Fill-in has failed.

Points that can be improved in the next time： 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. 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. 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.