作者asd235s89 (Alarm Error)
看板Biotech
标题[求救] Circular template Ligation
时间Mon Jun 6 23:43:54 2022
各位学长姐好:
有关以DNA Sequence合成圆形模板及纯化方式的问题，想与前辈们请益
我将自行黏合好的Circular template进行RCA环滚扩增反应後，
在agarose gel上有明显的亮带。
但不加primer时，却也有亮带产生。
理论上，without primer的情况下不会有RCA product 产生，
但有用外切酶反应，dPAGE纯化时也只看到一个band，
是没有纯化乾净吗? 还是试剂之类的有污染?
这部分卡了三个月，麻烦学长姊们提点，感激不尽
我的圆形模板合成步骤如下:
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序列设计无误)
合成完後纯化圆形模板的方式为: 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实验步骤:
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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