- Section 11 Exercise 11 6 Question 4 Homework Unanswered Consider The Samples Tested A Why Are The Samples Run In Tripl 1 (24.49 KiB) Viewed 218 times
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Section 11 Exercise 11-6 Question 5 Homework. Unanswered Refer to the procedural diagram in Figure 11.23 above for the following questions: a. In Step 4, what could be the consequence of washing too vigorously with wash buffer? Of not washing enough? b. What might be the consequence of not adding blocking agent in step 6? (Omit this question if blocking agent was not used.) c. In step 13, what would be the consequence of washing too vigorously with wash buffer? Of not washing enough? d. Instep 15, what would be the consequence of washing too vigorously with wash buffer? Of not washing enough?
899999999999 Step 1: Labeling the Microtiter Strip Using a permanent marker, label the outside of each well as shown. Replace numbers 1 and 2 with the "Patient Samplo" numbers you have been assigned PA 50 ML Stop 2: Adding Purified Antigen (Ag) Add 50 HL of purified antigen (green microtube labeled "PA) to each of the 12 wells, Incubato at your lab denk for 5 minutes. Dispone of the pipette tip properly, Stop 3: Remove Unbound Purified Antigon Gently tap the strip on several thickness of fresh paper towels to remove unbound antigen Be careful not to mix contents between wells. Dispose of wat towels. Wash Buffer
Wash Buffer Step 4: Washing the Wells Using the disposable transfer pipette, fil (but don't overfill and mix neighboring well contents) al 12 Wells with wash buffer. Save the disposable pipette for subsequent wash steps. Dispose of wet towels. (Note: In reality, at this point the entire lining of the well would be coated with Ag, but only two are shown to reduce cluttering in the art.) Step 5: Removing the Wash Buffer Drain the wells onto several thick- nesses of fresh paper towels, and then gently tap out the remainder. Do not allow mixing between wells. Dispone of wet towels. Repeat steps 4 and 5 to wash a second time. Continue with step 8 (optional) or step 10, 50 L BA XXXXXXXXX XXXXXX XXXXX Stop 6: Adding Blocking Agent (Optional) Add 50 pil of blocking agent (shown as x's in the drawing) to each weil. Incubate at your lab desk for 5 minutes Step 7: Romoving Unbound Blocking Agent Gently tap the strip on several thicknotes of fresh paper towels to remove unbound blocking agent. Be careful not to mix contents between wells.
Wush Butter TUOTE XXXXXX GER XXXXXXXXXX Stop & Washing the Wolle Uning the disposable transfer pipette, fill (but don't overfil and mix neighboring well contenta) al 12 Wolle with woh buffer. Step 3: Romoving the Wash Butfor Drain the welle onto several thicknesses of fresh paper towels. Do not allow mixing between welle. Tap out the last ronnants of wash buffer. Dispose of wet towels. Repeat steps 8 and 9 to wash a second time. 50 L 50 L XXXCO XXXXXXXXX XXXXXXXX XOOXXXXXX XXX XXXXXXXXXXX ккккккккккк XXXXXXXXXXX Stop 10: Adding the Positive Control Add 50 pl. of positive control (violet microtube labeled "+") to the three "+" wello. Continue immediately with step 11. Stop 11: Adding the Negative Control Add 50 ML of negative control (blue microtube labeled to the three "-" Wells. Continue immediately with step 12
50 L Wash Buffer XXXXXXX २८ XXXXXXXX XXXXXXX U? XXXXXXXXXXX XXXXXXXXXXX XXXXXXXXXX Step 12: Adding Patient "Serum Sampleo" Add 50 ML of Patient Step 13: Removing the Control and Patient Serum Sample Serum Sampleo" (yellow mlorotubes labeled with numbers) to the Solutions and Washing the Wolle Gently tap the strip on several appropriate remaining wells. Incubate for 5 minutos at your lab dook. thicknesses of fresh paper towels to remove the Control and "Patient The question marke indicate that the contents of the patient's Sample Serum Sample solution. Be careful not to mix contents between well are unknown. Perform two washes with wash buffer as shown in steps 8 and 9 The illustration shows what a positive result looks like at this point. A negative result would not have the antibody attached to the antigens and would look like the art in step 11
50 L SA Wash Buffer XXXXXXXX XXXXXXX XXXXXXXXXXX XXXXXXXXXXX (XXXXX XXXXXXXXXXX Stop 14: Adding Secondary (Conjugate) Antibody Add 50 L of secondary antibody (orange microtubes labeled with "SA") to the 12 wells. Incubate for 5 minutes at your lab desk. on several thicknesses of fresh paper towels to remove unbound sec- ondary antibody solution. Be careful not to mix contents between wolle. Perform three washes with wash buffer as shown in steps 8 and 9. The illustration shows what a positive result looks like at this point A negative result would not have either antibody present and would look like the art in step 11 50 L
Perform three washes with wash buffer as shown in steps 8 and 9. The illustration shows what a positive result looks like at this point. A negative result would not have either antibody present and would look like the art in step 11 50 L PR Step 16: Adding Substrate Add 50 pil of substrate (brown microtube labeled "SUB) to the 12 wolle, Incubate at your lob desk and road results within 5 minutos. A blue color in a positive result. Waiting longer than 5 minutes to read results could lead to the substrate turning blue through its exposure to light, not enzyme. 11.23 Procedural Diagram for the ELISA Pay special attention to volumes solutions, and pipettes used in each step Fresh pipette tips must be used for each solution and disposed of properly. The same disposable pipette should be used for transferring wash buffer throughout take care not to mix solutions from neighboring wells by overfilling or by emptying over a contaminated part of the paper towels