[求救] Circular template Ligation
各位學長姐好:
有關以DNA Sequence合成圓形模板及純化方式的問題，想與前輩們請益
我將自行黏合好的Circular template進行RCA環滾擴增反應後，
在agarose gel上有明顯的亮帶。
但不加primer時，卻也有亮帶產生。
理論上，without primer的情況下不會有RCA product 產生，
但有用外切酶反應，dPAGE純化時也只看到一個band，
是沒有純化乾淨嗎? 還是試劑之類的有汙染?
這部分卡了三個月，麻煩學長姊們提點，感激不盡
我的圓形模板合成步驟如下:
1. Mixing the Template DNA, Ligated DNA and water by the pipet.
2. Heat at 90°C for 5 minutes, cool at room temperature 10 minutes.
3. Add buffer and T4 ligase.
4. Incubate at room temperature for 2 hours. Deactive at 65°C for 20 mins.
5. Add ExoI, ExoIII and NEB buffer.
6. React at 37°C for 30 minutes, deactive at 80°C for 20 mins.
5. Ethanol precipitating for 6 hour under -20°C.
6. Remove solution and dry the product.
(以上提到的DNA序列設計無誤)
合成完後純化圓形模板的方式為: Purify with 10% dPAGE.
1. Pour solution into plate with rapid tapping to prevent bubbles.
2. Insert the comb until the gel is fully polymerized.
3. Remove the comb gently. Rinse the well with DI.
4. Put the gel into chamber. Pour the 1x TBE buffer into chamber and rinse
the well by syringe.
5. Loading sample and running under 200 V for 30 min.
6. Gel was visualized by UV shadow, mark band positions and slice the gel
into 1.5 mL tube, crush by tip.
7. Add 700 μL Elution Buffer, vortex under 1000 rpm for 20 min under RT.
8. Centrifuge under 12000 rpm for 10 min. Transfer 400 μL supernatant into
1.5 mL tube, add 5 μL 3 M pH 5.2 NaOAc and 1 mL EtOH. Stand under -20°C
for 8 hrs.
9. Centrifuge with 12000 rpm under -20°C , remove solution and rinse pellet
with chill 70% EtOH.
10. Centrifuge with 12000 rpm under -20°C , remove solution and dry the
pellet.
11. Re-suspend with DI and measure with nano-drop.
Linear RCA實驗步驟:
1. Mixing the circular template, primer, dNTP and water by pipette.
2. Heat at 90°C for 5 minutes, cool down for 10 minutes.
3. Add phi29 and buffer then mix by pipette.
4. Incubate at 25°C for 30 minutes. Heat deactivate at 90°C for 10 minutes.
5. Run 10 μL product + 2 μL 6X loading dye on 0.6% TAE agarose with 1X SYBR.
6. Run at 200 V for 30 min, and image by gel image system.
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