昨天和朋友出去玩
问他what’s the difference betwenn being a TA and being an instructor
他说当instructor就是，你要处理一些，你不知道为什么你要处理的事情，比如他的课要写很多report，然后有一个学生，对键盘过敏，所以不能打字，每次写report就要他反复和DSP(Disability Services and Programs)沟通来商讨解决方案
他：所以为什么学校要让这种学生来这种她根本不可能在之后取得career的专业？？？？
我：……
问他what’s the difference betwenn being a TA and being an instructor
他说当instructor就是，你要处理一些，你不知道为什么你要处理的事情，比如他的课要写很多report，然后有一个学生，对键盘过敏，所以不能打字，每次写report就要他反复和DSP(Disability Services and Programs)沟通来商讨解决方案
他：所以为什么学校要让这种学生来这种她根本不可能在之后取得career的专业？？？？
我：……
朋友：他的课他简直觉得大概只有20%的人可以pass，而且只是pass哦都不是说给高分。但是在这种该死的教育体系下，大家都维持了一个表面的体面。
我：真应该把你这些话给你学生说，我真不知道你rate my professor上面的5/5是怎么来的，尤其是什么“He is a generous with his grading”
我：真应该把你这些话给你学生说，我真不知道你rate my professor上面的5/5是怎么来的，尤其是什么“He is a generous with his grading”
我感觉我凉了
遇到隔壁lab的a，聊起b，结果我说错了b的名字，a给我纠正了，我说不好意思不好意思
然后遇到了b，聊起a，结果我说错了a的名字
完了他们一勾兑不就知道我是大傻逼了吗 可是记名字真的好难啊啊啊啊啊啊！！！
遇到隔壁lab的a，聊起b，结果我说错了b的名字，a给我纠正了，我说不好意思不好意思
然后遇到了b，聊起a，结果我说错了a的名字
完了他们一勾兑不就知道我是大傻逼了吗 可是记名字真的好难啊啊啊啊啊啊！！！
🥰2
For visualization, fluorescent probes were used at the following concentrations: 250nM Alexa488-, 568- or 647-tubulin, 40nM EB1-GFP, 10nM Kif4A-GFP, 20nM GFP-PRC1E, 20nM GFP-DasraA, and 0.1-1 μg/mL Alexa674-IgG against AurkB to visualize the CPC, Alexa568- IgG against a Kif23-CTP to visualize the Centralspindlin complex, Alexa568-IgG against a Ki20AE-CTP to visualize Kif20AE.
水水的摸鱼日常
140nM mCherry-rGBD to visualize RhoA-GTP
The Rhotekin fragment (rGBD) specifically binds to active, GTP-bound RhoA (16). Mouse Rhotekin gene was a gift from William Bement (University of Wisconsin–Madison, WI).
水水的摸鱼日常
老板要搞个清华的女生过来 看来老板已经山穷水尽了
甚至是个有master degree的人，来了我就不是本lab年龄最大的人了！！！！！希望老板赶紧骗过来！
12/7
1. F-actin using Lifeact-GFP: David Burgess (Boston College, MA)
2. RhoA: His-rGBD-mCherry. The Rhotekin fragment (rGBD) specifically binds to active, GTP-bound RhoA
The Rho GTPase-binding domain (rGBD) of mouse Rhotekin (amino acids 7-82) was subcloned into pGEX-4T-1 bacterial expression vector with an N-terminal 6xHis-mCherry tag, replacing the GST tag between EcoN1/Not1 restriction sites, creating mCherry-rGBD. Mouse Rhotekin gene was a gift from William Bement (University of Wisconsin–Madison, WI).
GFP-rGBD can be purchased online - https://www.addgene.org/26732/ (also from William Bement)
3. CPC: label its subunit DasraA
The X. laevis DasraA gene (IRBHp990G0393D, Source Bioscience, UK) was subcloned into pGEX-4T-1 with an N-terminal 6xHis-eGFP tag, creating GFP-DasraA.
12/7
Using PEG-passivated coverslips, we saw no recruitment of RhoA-GTP. Using lipid bilayers, the RhoA-GTP reporter was enriched at the bilayer under AAIZs.
1. F-actin using Lifeact-GFP: David Burgess (Boston College, MA)
2. RhoA: His-rGBD-mCherry. The Rhotekin fragment (rGBD) specifically binds to active, GTP-bound RhoA
The Rho GTPase-binding domain (rGBD) of mouse Rhotekin (amino acids 7-82) was subcloned into pGEX-4T-1 bacterial expression vector with an N-terminal 6xHis-mCherry tag, replacing the GST tag between EcoN1/Not1 restriction sites, creating mCherry-rGBD. Mouse Rhotekin gene was a gift from William Bement (University of Wisconsin–Madison, WI).
GFP-rGBD can be purchased online - https://www.addgene.org/26732/ (also from William Bement)
3. CPC: label its subunit DasraA
The X. laevis DasraA gene (IRBHp990G0393D, Source Bioscience, UK) was subcloned into pGEX-4T-1 with an N-terminal 6xHis-eGFP tag, creating GFP-DasraA.
12/7
Using PEG-passivated coverslips, we saw no recruitment of RhoA-GTP. Using lipid bilayers, the RhoA-GTP reporter was enriched at the bilayer under AAIZs.
www.addgene.org
Addgene: GFP-rGBD
Plasmid GFP-rGBD from Dr. William Bement's lab contains the insert rhotekin and is published in J Cell Biol. 2005 Jan 31. 168(3):429-39. This plasmid is available through Addgene.