该协议描述了如何使用PhyB和PIF3进行光遗传学实验以控制红光和远红光的基因表达。其中包括构建简单灵活的照明系统的分步说明,该系统可以使用计算机控制基因表达或其他光遗传学。
使用光控制生物过程提高了研究人员操纵许多生物过程的准确性和速度。光学控制允许前所未有的解剖功能的能力,并具有实现新型基因疗法的潜力。然而,光遗传学实验需要足够的光源,具有空间、时间或强度控制,这通常是研究人员的瓶颈。在这里,我们详细介绍了如何构建低成本和多功能的LED照明系统,该系统可针对不同的可用光遗传学工具轻松定制。该系统可配置为手动或计算机控制,LED强度可调。我们提供了一个图解的分步指南,用于构建电路,使其由计算机控制,并构建LED。为了便于该器件的组装,我们还讨论了一些基本的焊接技术,并解释了用于控制LED的电路。使用我们的开源用户界面,用户可以在个人计算机(PC)或廉价平板电脑上自动执行精确的计时和光脉冲。这种自动化使该系统可用于使用LED控制基因,信号通路和其他跨越大时间尺度的细胞活动的实验。对于该协议,不需要电子学方面的先验专业知识来构建所需的所有部件或使用照明系统进行光遗传学实验。
光遗传学工具正变得无处不在,并且不断开发新技术来光学控制生物过程,例如基因表达,细胞信号传导等等1,2,3。用光控制细胞过程的能力允许快速动力学、严格的空间控制和剂量依赖性调节,这些调节可以通过光强度和曝光时间来控制。要使用这些工具,需要控制这些参数的设备。我们最近开发了一种基因编码的PhyB-PIF3哺乳动物基因开关,它分别使用红光/远红光可逆地激活和失活基因4。该系统在几种哺乳动物细胞系中进行了测试,即使在非常少量的光(包括光脉冲)下也能实现无与伦比的基因表达诱导。希望使用PhyB开关和类似工具5,6的研究人员经常请求有关控制照明强度和持续时间的方法的信息。因此,我们开发了该协议,并附有分步说明,以便更广泛地采用这些光遗传学工具。
在LED广泛使用之前,带有滤光片的宽带光源用于研究光敏色素7等光敏蛋白。最近,一些LED照明系统已经与光遗传学工具8,9,10,11,12一起发布,但这些协议可能需要电子/软件方面的大量专业知识,需要专门的设备(例如,3D打印机,激光切割机或光掩模),或者没有提供一些研究人员需要部署的分步说明。虽然独立控制多孔板中的单个孔可能很有用,但当研究人员只需要在明光和暗光或红光与远红光下比较几个不同的样品时,通常没有必要。此外,许多现有的商业系统价格昂贵,定制能力有限。但是,该协议中描述的LED具有成本效益,明亮,并且可以以多种方式安装;因此,它们可用于照亮几种不同类型的样品。通过提供的协议和软件,可以使用和控制从紫外 (UV) 到 NIR 的 LED,以使用 UVR8 13,14、Dronpa15,16、LOV 域 17,18、阶跃函数视蛋白 19,20、CRY2 21,22、PhyB4,23,24 进行光遗传学实验,25、细菌光敏色素26、27、28、29等光响应系统30、31、32。
该协议构成了控制光刺激不同参数所需的电路和其他硬件的组装教程,以及运行光遗传学实验的分子/细胞工具。此外,我们报告了从Kyriakakis等人4优化的质粒,这些质 粒更小,更稳定,可用于克隆。通过该协议,没有电子和光学专业知识的生物学家可以构建灵活而强大的照明系统。我们逐步展示了如何构建LED系统,消除了更广泛采用光遗传学工具的技术瓶颈。该系统可轻松用于大多数细胞培养箱,即使它们不包含线端口。例如,我们将 LED 系统连续保存在加湿的 CO2 培养箱中超过 6 个月,性能没有下降。我们还解释了如何将 LED 系统连接到计算机,并将其与我们在 GitHub 上提供的开源软件连接 (https://github.com/BreakLiquid/LED-Control-User-Interfaces)。使用此协议构建系统可为研究人员提供调试潜在问题、更换部件和改进/扩展功能的基本知识。
系统概述
构建照明系统涉及 (1) 构建电子电路,(2) 构建外围设备(电源线、电源开关等),(3) 构建 LED,(4) 组装所有这些组件,以及 (5) 安装软件以通过用户界面控制 LED(图 1A)。完成后,照明系统可以通过用户界面独立控制多达四个 LED(图 1B)。用户界面使每个 LED 能够以指定的时间间隔脉冲,并在指定时间后关闭。还有一个开始延迟,可以在指定时间开始照明程序。电位计 (POT) 可独立调节每个 LED 的强度,也可用于无需计算机即可手动控制 LED。LED的电线可以是任何自定义长度,使其可以轻松放置在培养箱或实验室空间中。由于这些LED的高功率,它们可用于从远处用单个LED照亮大面积区域。
指示灯驱动器说明
为了给LED供电和控制强度,该协议将通过构建“LED驱动器”的步骤。每个 LED 都有一系列工作电压(图 1C)。在工作期间,控制光强度的稳压器的输出电压可通过电位计调节。POT改变电阻,调节输出电压/亮度。使用1kΩ(1千欧)电位器调谐可提供我们所说的“高压电路”,范围为1.35 V至2.9 V。 由于2.9 V对于操作低压LED来说太高(图1C),我们显示了单个修改(电阻3或“R3” 补充图1A),限制了与低压LED匹配的范围。当与电位计并联时,R3用于将施加到LED的最大电压降低到1.85 V( 补充图8中详述的组件)。通过使用电压而不是电流来控制亮度,该系统对于具有不同工作电压的LED更加灵活。 图1C 包含高压和低压LED列表,以指导最佳电路选择。这种设计将最小电压保持在足够低的水平,以便在电位计关闭时LED完全关闭,并且不允许电压超过LED的典型工作电压。对于PhyB光遗传学,我们使用深红色和远红色LED,它们使用低压电路。
LED电脑控制系统说明
LED照明系统可用于无需计算机或微控制器的恒定照明。但是,对于脉冲程序和控制单个LED定时,必须安装微控制器。要使用微控制器来控制LED,需要一个晶体管将微控制器连接到电路。该晶体管感测来自微控制器的电压,并从导电或绝缘切换。为了控制“开”和“关”,我们使用所谓的“NPN开关型晶体管”(2N2222)作为R2上的可控分流器(补充图1A)。当来自微控制器的电压施加到晶体管基极时,晶体管变得导电并使LED电压变低,从而关闭LED。因此,LED和晶体管的开启和关闭状态由微控制器直接控制,微控制器由PC上安装的软件控制。
要制作照明系统,需要以下步骤:构建电路;构建电源、手动电源开关、POT 和微控制器连接;构建发光二极管;容纳一个黑匣子以适应照明系统;连接所有布线和设备;安装LED控制软件,用光刺激细胞;使用双荧光素酶测定法测量基因表达。
这里描述的LED系统已被我们的实验室用于优化,表征和使用几种光遗传学工具。在Kyriakakis等人4中,我们并行测试了许多PhyB-PIF基因开关的组合。然后,我们使用该系统测试不同频率的光脉冲,以测量基因开关动力学和有效光强度。该系统还用于优化和表征两个使用蓝光进行刺激的光遗传学系统5,6。由于只需要一个LED足够亮才能激活大多数光遗传学工具,因此并不总是需要购买每个孔上具有大量LED的系统。这种设置价格低廉、可靠、易于重新配置,并且不需要事先的电气专业知识即可遵循组装协议。
在补充 补充图31–38中,我们描述了如何将多达四个LED集成到系统中。虽然这可能会限制一些需要大量并联条件的实验,但可以通过用更高瓦数的电源替换该协议中使用的 9 伏电源来添加更多 LED。同样,几个较低功率的LED可以并联到每个电路。在后一种安排中,某些LED不会单独控制,但是当需要许多LED覆盖更大的区域时,这可能很有用。一旦熟悉了该系统的电子设备,就有很多方法可以对其进行定制。定制系统的其他策略包括将LED放置在离样品更远或更近的地方,并通过滤光片/扩散器照明均匀的照明条件或防止加热,如(补充图23)和Allen等人5所示。我们 LED 设计的另一个显着特点是它封装在环氧树脂中,背面有一个触摸紧固件;这使得 LED 几乎可以轻松安全地放置在任何地方:孵化器、鱼缸、动物笼子、墙壁等。
许多使用光遗传学来控制基因、信号通路和其他细胞活动的实验通常需要脉冲、跨越大时间尺度或需要在培养箱中进行,因此需要在没有显微镜的情况下进行自动化或远程操作。该 LED 系统已在加湿的 CO2 培养箱内连续测试了几个月,没有任何问题。此外,对于可逆系统(如PhyB光遗传学系统),实验者可能需要对特定的脉冲照明时间表进行编程。在我们之前的工作4中,我们使用脉冲程序通过用户界面测试哺乳动物细胞中PhyB-PIF3开关的可逆性动力学。使用本手稿中描述的方法,对脉冲协议进行编程很容易,以用户友好的方式提供许多类型的光遗传学实验所需的灵活性和自主性。
构建该系统的最关键步骤包括将电路放在PCB板上并连接组件,详见第1节和第2节。在焊接每个组件之前,必须仔细遵循这些部分中的每个步骤,并逐行仔细检查针孔编号。第2节介绍如何设置将连接到电路的组件。为了使组件以正确的方向连接,确保线对线连接器上的黑色和红色电线的颜色匹配尤为重要。这两个部分中的小疏忽很可能会影响系统的功能。实际上,对此方法进行故障排除的第一步是检查电路是否正确构建以及所有连接是否到位。其次,检查连接松动的焊接质量以及可能使电路短路的扩口线毛的导线尤为重要。第三步是确保 LED 正常工作,这可以使用电源或 1.5 V 电池来完成,方法是用鳄鱼夹夹住 LED 的两个端子。另一个潜在的关键考虑因素是防止加热(在高功率下使用LED时)或漫射光以获得更广泛的照明。为了解决这些考虑因素,LED可以安装在黑匣子外面,内部有“隐私膜”,如 补充图23 和Allen等人5所述。由于该系统的简单性,将其拆开以验证、修改、升级或修复模块化组件并不困难。
诱导基因系统的另一个关键因素是考虑需要多少激活或被控制的生物系统可以接受多少泄漏。如图 6所示,这些可以随报告基因DNA的数量而变化。此外,转染效率以及每个细胞中报告基因构建体的拷贝数也会有所不同。对于某些实验来说,制造具有固定量的报告基因或PhyB基因转换组分的细胞系并筛选具有所需诱导表达范围的克隆可能是有利的,就像药物诱导系统通常所做的那样。由于慢病毒质粒pPK-2304的大小和不稳定性,我们还在pcDNA骨架pPK-351(Addgene #157921)和pPK-352(Addgene #157922)中制作了PhyB开关的非慢病毒质粒版本。
通过按照该协议构建此LED照明系统,用户拥有在体外和体内进行各种光遗传学实验所需的所有组件。结合在哺乳动物细胞中使用PhyB-PIF3的说明,该协议将允许非工程师和生物学家灵活有效地在各种情况下使用基于PhyB的光遗传学。
The authors have nothing to disclose.
我们要感谢Yingxiao (Peter) Wang,Ziliang Huang和Molly Allen在开发过程中测试了不同版本的LED系统。这项工作得到了加州大学圣地亚哥分校Kavli脑与心研究所和索尔克研究所,国家科学基金会通过NSF信息科学中心在Grant CCF-0939370,NIH Grant NS060847和NIH Grant R21DC018237下的支持。
18AWG 2pin RED Black wire | Amazon | 15M-28AWG-2468 | Inexpensive wire to connect LEDs to the power circuit. https://www.amazon.com/gp/product/B072KGYH1M/ref=oh_aui_detailpage_o05_s00?ie=UTF8&psc=1 |
1K Ohm potentiometer | Amazon | 52161500 | 2 x 1K Ohm potentiometer potential + 2 x black control Knob. https://www.amazon.com/gp/product/B00XIWA2GO/ref=oh_aui_detailpage_o00_s00?ie=UTF8&psc=1 |
20 Gauge Silicone JST Connector | Amazon | SIM&NAT 5.9 inch 2 Pin Male Female JST RCY Plug Connectors | These are very common and there are many equivalents. https://www.amazon.com/gp/product/B071XN7C43/ref=oh_aui_detailpage_o00_s01?ie=UTF8&psc=1 |
22 AWG solid jumper wires | Amazon | WJW-60B-R | Jameco Valuepro WJW-60B-R Wire Jumper Kit 350 each 22 AWG, 14 Lengths 10 Colors 25 Of Each Length. https://www.amazon.com/Jameco-Valuepro-WJW-60B-R-Jumper-Lengths/dp/B01KHWEB3W/ref=sr_1_5?s=industrial&ie=UTF8&qid=1519261370&sr=1-5&keywords=solid+wire+breadboard&dpID=51UopZhPJeL&preST=_SX342_QL70_&dpSrc=srch |
560 ohm 1/2watt 1% tolerance | Amazon | a14051600ux0301 | Uxcell a14051600ux0301 60 Piece Axial Lead 1% Tolerance Colored Ring Metal Film Resistor Resistance, 560 Ohm 1/2W. https://www.amazon.com/a14051600ux0301-Tolerance-Colored-Resistor-Resistance/dp/B016ZU2DGC/ref=pd_day0_328_9?_encoding=UTF8&pd_rd_i=B016ZU2DGC&pd_rd_r=XTM6KHQ3NT8DHWB1QWZN&pd_rd_w=txGNx&pd_rd_wg=ELyii&psc=1&refRID=XTM6KHQ3NT8DHWB1QWZN |
820 ohm 1/2watt 1% tolerance | Amazon | TTL-A-8035-50Ea | Set of 50Ea Metal Film Resistor 820 Ohm 1% 1/2W (0.5W). https://www.amazon.com/50Ea-Metal-Film-Resistor-0-5W/dp/B00VGU2SS0/ref=sr_1_14?s=industrial&ie=UTF8&qid=1518045187&sr=1-14&keywords=1%2F2W+820+Ohm+resistor |
A Male to B Male Cable (10 Feet) | Amazon | Part# 30-001-10B | The cable that comes with the Arduino doesn't fit well in the box. https://www.amazon.com/gp/product/B001MSU1HG/ref=oh_aui_detailpage_o07_s00?ie=UTF8&psc=1 |
Ardiuino UNO equivilent | Amazon | Elegoo EL-CB-001 | UNO R3 Board ATmega328P ATMEGA16U2 with USB Cable for Arduino. https://www.amazon.com/gp/product/B01EWOE0UU/ref=oh_aui_detailpage_o03_s00?ie=UTF8&psc=1 |
Arduino holder | Digikey | X000018 | Fits very snug. https://www.digikey.com/product-detail/en/arduino/X000018/1050-1150-ND/8135632 |
Black boxes for circuits and light chambers | Amazon | 1591ESBK | Hammond 1591ESBK ABS Project Box Black. https://www.amazon.com/gp/product/B0002BSRIO/ref=oh_aui_detailpage_o07_s00?ie=UTF8&psc=1 |
Blue LED | Digikey | LXML-PB01-0040 | LED LUXEON REBEL BLUE SMD. Uses "Saber 20 mm Star base" https://www.digikey.com/product-detail/en/lumileds/LXML-PB01-0040/1416-1029-1-ND/3961134 |
Cable ties | Amazon | sd027 | Tarvol Nylon Zip Ties (Pack of 100) 8 Inch with Self Locking Cable Ties (White). https://www.amazon.com/Tarvol-Nylon-Locking-Cable-White/dp/B01MRD0JRR/ref=sr_1_7?s=hi&ie=UTF8&qid=1519261882&sr=1-7&keywords=Cable+ties&dpID=51zUNmuUjyL&preST=_SY300_QL70_&dpSrc=srch |
Command Fridge Clips | Amazon | 17210CLR | Clips for holding circuit board inside of the black box. Command strips can also be used. https://www.amazon.com/gp/product/B0084M69YM/ref=oh_aui_detailpage_o00_s01?ie=UTF8&psc=1 |
Cyan LED | Digikey | LXML-PE01-0070 | LED LUXEON REBEL CYAN SMD. Uses "Saber 20 mm Star base" https://www.digikey.com/products/en?keywords=1416-1031-1-nd |
Electrical tape – 3M Scotch #35 Electrical Tape Value Pack | Amazon | 03429NA | Scotch 700 Electrical Tape, 03429NA, 3/4 in x 66 ft. https://www.amazon.com/Scotch-Electrical-Tape-4-Inch-66-Foot/dp/B001ULCB1O/ref=psdc_256161011_t1_B001B19FDK |
Farred LED 720nm | Luxeon Star LEDs | LXML-PF01 | Far Red (720nm) LUXEON Rebel LED. Uses "Saber 20 mm Star base" https://www.luxeonstar.com/lxml-pf01-far-red-luxeon-rebel-led-260mW |
Farred LED 740nm | Ushio | EDC740D-1100-S5 | Uses "STAR XP 3535" base https://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC740D-1100-S5.pdf |
Farred LED 780nm | Ushio | EDC780D-1100 | Uses "STAR XP 3535" base http://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC780D-1100.pdf |
Farred LED 810nm | Ushio | EDC810D-1100 | Uses "STAR XP 3535" base http://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC810D-1100.pdf |
Farred LED 850nm | Ushio | EDC850D-1100 | Uses "STAR XP 3535" base http://www.ushio-optosemi.com/jp/products/led/power/pdfs/edc/EDC850D-1100.pdf |
Grommets | Amazon | Pico 6120D | These are very common and there are many equivalents. https://www.amazon.com/Pico-6120D-Vinyl-Grommets-Package/dp/B0002ZG47G |
Hair/Alligator Clips | Amazon | 1-3/4 Inch (45 Mm)- Hair Clips Single Prong Metal Alligator Clips Hairbow Accessory -Silver,50 Pcs. https://www.amazon.com/gp/product/B00K09T3L8/ref=oh_aui_detailpage_o00_s00?ie=UTF8&psc=1 |
|
LED base | Luxeon Star LEDs | LXB-RS20A | Saber 20 mm Star Blank Aluminum MCPCB Base For Rebel LEDs https://www.luxeonstar.com/saber-20mm-star-blank-mcpcb-base-for-a-rebel-leds |
LED PCB fopr Ushio LEDs | Adura LED solutions | STAR XP 3535 Package LED | Fits many other LEDs by Ushio http://aduraled.com/product/pcb/1901-star-xp-3535-package-led |
Loctite Epoxy Clear Multi-Purpose, 0.85-Fluid Ounce Syringe | Amazon | 1943587 | Loctite Epoxy Clear Multi-Purpose, 0.85-Fluid Ounce Syringe. https://www.amazon.com/Loctite-Multi-Purpose-0-85-Fluid-Syringe-1943587/dp/B011INNBN0/ref=psdc_256243011_t4_B0044FBB8C |
NTE Heat Shrink 2:1 Assorted Colors and Sizes 160 PCS | Amazon | B000FIDTYG | These are very common and there are many equivalents. https://www.amazon.com/NTE-Heat-Shrink-Assorted-Colors/dp/B000FIDTYG/ref=sr_1_1?s=hi&ie=UTF8&qid=1519261282&sr=1-1&keywords=nte+shrink&dpID=41L5l7LCfiL&preST=_SX342_QL70_&dpSrc=srch |
Picture Hanging Velcro Strips | Amazon | PH204-16NA | With these you can hang the LEDs in many places. https://www.amazon.com/Command-Picture-Hanging-16-Pairs-PH204-16ES/dp/B073XS3CHV/ref=pd_sim_60_5?_encoding=UTF8&pd_rd_i=B073XS3CHV&pd_rd_r=112KGQJNRRYD0RAT598H&pd_rd_w=3n34Q&pd_rd_wg=sRvec&psc=1&refRID=112KGQJNRRYD0RAT598H |
Power supply | Amazon | tb013 | Any other 9V 1.5Z AC/DC converter will do becuase we cut the end off anyway. https://www.amazon.com/gp/product/B06Y1LF8T5/ref=oh_aui_detailpage_o07_s01?ie=UTF8&psc=1 |
Power switch Rocker Switch | Amazon | SIXQJZML | These are very common and there are many equivalents. https://www.amazon.ca/COOLOOdirect-Solder-Rocker-Switch-Toggle/dp/B071Y7SMVQ/ref=sr_1_31?_encoding=UTF8&c=ts&dchild=1&keywords=Boat+Rocker+Switches&qid=1594434474&s=sports&sr=1-31&ts_id=2438617011 |
Rectangular Connectors – for crimped wires | Digikey | 2183-1905-ND | 6 Rectangular Connectors – Housings Black 0.100" (2.54mm) https://www.digikey.com/product-detail/en/pololu-corporation/1905/2183-1905-ND/10450382?utm_adgroup=Rectangular%20Connectors%20-%20Housings&utm_source=google&utm_medium=cpc&utm_campaign=Shopping_Product_Connectors%2C%20Interconnects_NEW&utm_term=&utm_content=Rectangular%20Connectors%20-%20Housings&gclid=Cj0KCQjwvIT5BRCqARIsAAwwD-QmETT-ko07ote5VQgodKvWU0uDG8GYN7Vj-6WVBBOWdSgPaPd9azAaAhVLEALw_wcB |
Red LED | Digikey | LXM3-PD01 | LED LUXEON REBEL DEEP RED SMD. Uses "Saber 20 mm Star base" https://www.digikey.com/products/en?keywords=1416-1701-1-nd |
Sandpaper | Amazon | B002NEV6GS | 3M Wetordry Sandpaper, 03022, 800 Grit, 3 2/3 inch x 9 inch. https://www.amazon.com/3M-03022-Imperial-Wetordry-Sandpaper/dp/B002NEV6GS/ref=sr_1_1?dchild=1&keywords=3M+Imperial+Wetordry+3-2%2F3+in.+x+9+in.+800+Grit+Sandpaper+Sheets+%2810+Sheets-Pack%29&qid=1594435012&sr=8-1 |
Solder for soldering wires and circuit components | Amazon | Mudder Lead Free Solder Wire Sn99 Ag0.3 Cu0.7 with Rosin Core for Electrical Soldering 0.22lbs (0.6 mm) | These are very common and there are many equivalents. https://www.amazon.com/Mudder-Solder-Electrical-Soldering-0-22lbs/dp/B01B61TWGY |
Solder-able Breadboard for building the circuit | Amazon | GK1007 | Gikfun Solder-able Breadboard Gold Plated Finish Proto Board PCB Diy Kit for Arduino (Pack of 5PCS) GK1007. https://www.amazon.com/gp/product/B071R3BFNL/ref=oh_aui_detailpage_o04_s00?ie=UTF8&psc=1 |
Spade drill bit | Amazon | Irwin 88811 | These are very common and there are many equivalents. https://www.amazon.com/Speedbor%C2%AE-Blue-GrooveTM-Standard-Length-Woodboring/dp/B017S9JXB2/ref=sr_1_10?ie=UTF8&qid=1519516560&sr=8-10&keywords=11%2F16+spade+bit |
Transistor | Newark | 2N2222A | Can buy from many places. http://www.newark.com/nte-electronics/2n2222a/bipolar-transistor-npn-40v-to/dp/10M4197 |
Voltage regulator | Newark | LM317T | Equivilent to NTE956. https://www.newark.com/stmicroelectronics/lm317t/adjustable-linear-regulator-1/dp/89K0685?gclid=CjwKCAiAu9vwBRAEEiwAzvjq-1rmUi6lvOIFFt-BxttHXvmAeUoni4NM0BW-BtM_LMliSqxA9Xq4KxoCfikQAvD_BwE&mckv=sQqHZDDRz_dc|pcrid|219869297712|plid||kword|lm317t|match|p|slid||product||pgrid|35966450488|ptaid|kwd-541160713|&s_kwcid=AL!8472!3!219869297712!p!!g!!lm317t&CMP=KNC-GUSA-SKU-MDC |
Windows 10 tablet | Amazon | B08BYTT79Y | Any Windows 10 PC will do. https://www.amazon.com/gp/product/B08BYTT79Y/ref=ppx_yo_dt_b_asin_title_o01_s00?ie=UTF8&psc=1 |
Cell Culture Reagents | |||
Human Embryonic Kidney 293 cells HEK293 | ATCC | ATCC CRL-1573 | Common Cell line. https://www.atcc.org/products/all/CRL-1573.aspx |
Fetal Bovine Serum | ThermoFisher | 26140079 | These are very common and there are many equivalents. https://www.thermofisher.com/order/catalog/product/26140079#/26140079 |
Dulbecco’s Modified Eagle Medium High Glucose | ThermoFisher | 11965−092 | These are very common and there are many equivalents. https://www.thermofisher.com/order/catalog/product/11965118?SID=srch-srp-11965118#/11965118?SID=srch-srp-11965118 |
10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin | ThermoFisher | 15140122 | These are very common and there are many equivalents. https://www.thermofisher.com/order/catalog/product/15140122?SID=srch-srp-15140122#/15140122?SID=srch-srp-15140122 |
White Corning 96-Well Solid Black or White Polystyrene Microplates | ThermoFisher | 07-200-589 | White plates are preferred. Do not use clear plates. https://www.fishersci.com/shop/products/costar-96-well-black-white-solid-plates-8/p-152852 |
PEI MAX – Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000) | PolySciences | 24765-1 | Can be replaced with another transfection reagent. https://www.polysciences.com/default/catalog-products/life-sciences/transfection-reagents/polyethylenimine-max-mw40000-high-potency-linear-pei/ |
Name of Equipment | |||
Diagonal Cutting Plier (110mm) | Amazon | Proskit 1PK-037S | These are very common and there are many equivalents. https://www.amazon.com/iExcell-Diagonal-Cutting-Nippers-Chrome-Vanadium/dp/B076XYVS6Y/ref=sr_1_11?dchild=1&keywords=diagonal+cutting+pliers&qid=1594436230&sr=8-11 |
Dremil 3000 with cutting tool and grinder | Amazon | Dremel 3000 | Dremel 3000-2/28 Variable Speed Rotary Tool Kit- 1 Attachments & 28 Accessories- Grinder, Sander, Polisher, Router, and Engraver. https://www.amazon.com/Dremel-3000-2-28-Attachments-Accessories/dp/B005JRJE7Y/ref=sr_1_3?dchild=1&keywords=Dremel+200-1%2F15+Two-Speed+Rotary+Tool+Kit&qid=1594436404&s=hi&sr=1-3 |
Dremil cutting and grinding tool | Amazon | Dremel 200-1/15 | Any similar Dremil will work. https://www.amazon.com/Dremel-200-1-Two-Speed-Rotary-Tool/dp/B002BAHF8W/ref=sr_1_1?s=hi&ie=UTF8&qid=1519268058&sr=1-1&keywords=dremel+200&dpID=41h9ZucnTYL&preST=_SY300_QL70_&dpSrc=srch |
Dremil grinding tip | Amazon | Dremel 84922 | Silicon Carbide Grinding Stone. https://www.amazon.com/Dremel-84922-Silicon-Carbide-Grinding/dp/B00004UDKD/ref=sr_1_fkmr0_1?s=hi&ie=UTF8&qid=1519268585&sr=1-1-fkmr0&keywords=dremel+tip+84922 |
EDSYN The Original Deluxe SOLDAPULLT | Amazon | DS017 | For removing solder/mistakes. https://www.amazon.com/EDSYN-The-Original-Deluxe-SOLDAPULLT/dp/B006GOKVKI |
Helping Hand with Magnifying Glass | Amazon | SE MZ101B | These are very common and there are many equivalents. https://www.amazon.com/SE-MZ101B-Helping-Magnifying-Glass/dp/B000RB38X8/ref=sr_1_4?s=hi&ie=UTF8&qid=1519268108&sr=1-4&keywords=Helping+hands&dpID=31GEhMw7WvL&preST=_SX300_QL70_&dpSrc=srch |
Pointed Nose Micro Pliers | Amazon | Hakko CHP PN-20-M | Steel Super Specialty Pointed Nose Micro Pliers with Smooth Jaws, 1.0mm Nose. https://www.amazon.com/Hakko-PN-20-M-Specialty-Pointed-Pliers/dp/B00FZPGUBI/ref=sr_1_1?s=hi&ie=UTF8&qid=1519268153&sr=1-1&keywords=Hakko+CHP+PN-20-M+Steel+Super+Specialty+Pointed+Nose+Micro+Pliers+with+Smooth+Jaws%2C+1.0mm+Nose&dpID=3109XRgwn3L&preST=_SX342_QL70_&dpSrc=srch |
Small screw drivers | Amazon | Wiha 26197 | These are very common and there are many equivalents. https://www.amazon.com/26197-Precision-Slotted-Phillips-Screwdrivers/dp/B01L46TEN2/ref=sr_1_1?s=hi&ie=UTF8&qid=1519268018&sr=1-1&keywords=Wiha+precision+set |
Soldering iron | Amazon | Yihua 939D+ Digital Soldering Station | These are very common and there are many equivalents. https://www.amazon.com/Professional-Digital-Soldering-Station-Switch/dp/B07YSCBZ4F/ref=psdc_13837391_t1_B07RVMZNYR |
TraceTech No-Clean Flux Pen | Amazon | 2507-N | Tech Spray 2507-N No-Clean Flux Dispensing Pen, 11.5 mL. https://www.amazon.com/Tech-Spray-2507-N-No-Clean-Dispensing/dp/B00DDF2FYS/ref=sr_1_1?dchild=1&keywords=2507-N&qid=1595469618&sr=8-1 |
Weller WSA350 120v Bench Top Smoke Absorber | Amazon | WSA350 | For soldering safety. https://www.amazon.com/Weller-WSA350-Bench-Smoke-Absorber/dp/B000EM74SK |
Wire strippers | Amazon | CSP-30-7 | These are very common and there are many equivalents. https://www.amazon.com/Hakko-CSP-30-7-Stripper-Maximum-Capacity/dp/B00FZPHY7M/ref=psdc_553398_t5_B00FZPHMUG |
IWISS IWS-3220M Micro Connector Pin Crimping Tool 0.03-0.52mm² 32-20AWG | Amazon | IWS-3220M | These are very common and there are many equivalents. https://www.amazon.com/gp/product/B078WPT5M1/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&psc=1 |