在样品池中插入温度传感器的PAC-743帕耳帖样品池转换器的DNA熔化测量

对于热熔研究，可以将温度传感器插入样品池中，并绘制样品的实际温度，以提高熔融实验的温度读数的准确性。这种测量技术很容易应用于具有较大样品体积的10mm矩形样品池。然而，对于样品体积较小（<100µL）的样品，温度传感器探针会阻挡仪器的光路。然后很难同时获得样品的吸光度和温度测量值。通过使用其中一个样品池来监测样品温度而不获得该位置的吸光度测量值，可以准确地记录样品溶液的熔化温度。此外，当对极少量可用的样品进行熔融测量时，样品可能在高温下挥发，这经常使分析过程复杂化。在样品池上放置一个硅帽可以防止样品挥发。Application NoteUV-0016-17 DNA Melting Measurements using the PAC-743 Peltier CellChanger with a Temperature Sensor Inserted in the Cell2/4Application Note DNA Melting Measurements using the PAC-743 Peltier Cell Changer with a Temperature Sensor Inserted in the Cel l In troduc t ion Thi s appl i cat i on note compares thermal melt measu r eme n ts of a DNA sam p le using the temperature sensor i nside a cel l a n d us i ng the PAC-743's holde r sensor. The 8-posit i on m i cro cell sensors as a t emperature mon i tor (Figure 1)， the horizontal axis of the temperature co u rse data can be plotted with actual t empe r atures obtained by the se n sor. T h is i ncreases the tempe r ature accuracy of small volume sample measurements wit h the 8-posit i on micro cel l. V-630 For thermal melt studies， a temperatu r e senso r can be inserted i n to the sample ce ll s and the actual temperatures of samples plotted i n order to inc r ease the accuracy of the UV-Vis Spectro p hotometer temperature readings for the melting experiment. This measurement tec h nique i s easily applied to 10 mm rectangular cells with larger sample volumes. Howeve r ， f or samples with small sample volumes (<100 p L) a temperature sensor probe blocks the in strument's opt i cal path. It is then difficult to obtain both absorbance and temperature measurements of a sample simultaneously. By usi n g one of the cells to mon i tor the sam p le temperature without obta ini ng absorbance meas u rements at that positio n ， the melt i ng temperature of t h e sample solut i on can be accurately recorded. Additionally， when performing a melting measurement o n samples available in extremely small amounts， volati l ization of the sample ca n occur at high temperatures， frequent l y complicating the analysis process. P l acing a si li con cap on the cell can prevent sample volatilizat i on. Figure 1. PAC-743 Pel t ier 8-posi t ion c ell c h ang er . K eywo r ds V-630，UV-Visible/NIR， Biochemist r y， PAC-743 water-cooled Pelt i er cell changer， VWTP-780 Temperature Control Meas u rement Prog r am Experimen t al A 20 pg/mL solu ti on of poly (dA-dT )-Poly (dA-dT) wa s made in KH2PO4-NaOH b uf f er at pH 7. Samples were measured in ce l ls 1-7 whi l e ce l l 8 was used on l y to mo n itor the sam p le temperature. Wavelength 260nm Response Fast Ramp Rate 2°C/min Start Condition ±0.01°C for 3 seconds Data Interval Results The melt in g curves f rom the results of s a mp l e measurements wi t h al l eight m ic r o cells us in g the hold e r se n sor a r e plotted as shown in Figure 2. No vo l at ili z ation of the sampl e s wa s observed (Figure 3). Figure 2. T h er m al mel t of p ol y (d A-d T )-Pol y (d A-dT ) usi n g the P AC-743 h older se n s or . Figure 3. C ha ng e in t h e sa mp l e v ol ume b e f or e a n d af t er ther m al m ea su r ements . I n order to enhance the accuracy of the temperatu r e， one of the eig h t cells (referred to as "cell 8") was used exclusively to mo n itor the sample temperature. F i gu r e 4 shows a result of melting curves using the temperature readings from i nternal cell se n sor. These temperature va l ues were plotted i n the horizontal axis in Figure 4 using data collected from internal cel l sensor i n cell 8. No evaporation was obse r ved in this case (Figure 5). 28600 Mary's Cour t ， E a s to n ， M D 21601US A UASGO T el ： (800) 333-5272， Fa x ： (410) 822-7526 A ppli c a tio n Lib r ary： htt p：//www.jas co in c .c o m/a pp l icatio n s Figure 4. Th er ma l mel t o f p o l y (dA -d T )-Pol y (d A-d T ) u si n g t h e i nt e r n al ce l l s e n s or. Figure 5. Ch ang e in t h e sa mpl e v ol ume be f ore a n d a f t e r ther m al me a suremen t s. The results of the mel t ing points， calculated from the melting curves data in F i gure 2 and 4， are s h own in Table 1. The results us i ng the h older sensor are shown i n Table 1 ranging mel tin g temperatu r e between 66.0°℃~66.2°℃ (average 66.1℃) with standard deviation of 0.08°℃ and coeff i c i e n t of variance of 0.13%. On the other hand， the melt i ng temperatu r e results u sing the i n ternal cell sensor range between 63.6°℃~63.8°C (average 63.7°C) with standard deviation of 0.08℃and coeffic i ent of var i ance of 0.12%. Table 1. Re co r ded mel t i n g tempe r atures measured us i n g th e h o l der a n d i n ter n al cell se n so r s . Holder Sensor Internal Cell Sensor Cell 1 66.1 63.6 Cell 2 66.0 63.6 Cell 3 66.0 63.6 Cell 4 66.1 63.6 Cell 5 66.1 63.7 Cell 6 66.0 63.7 Cell7 66.2 63.7 Cell 8 66.2 63.7 Average 66.1 一 Standard Deviation 0.08 0.08 C.V. 0.13 0.12 JASCO INC. 28600 Mary's Cour t ， E a s to n ， M D 21601US A C onclusions The data show tha t the melting temperature using the holder sensor was approximately 3.5°C h igher tha n using the internal ce l l se n so r ， while both the standa r d deviation and coefficient of variance had no major d i fferences in thei r result. These results indicate that the holder sensor ca n su ff iciently measure a reproducible melting temperature for all sample cells. However ， to obtai n the absolute temperature va l ue for sample mel t s ， a cell tempe r ature sensor is h ighly recommended. 28600 Mary's Cour t ， E a s to n ， M D 21601US A