Andrew:Notebook/C1 InTube 150525: Difference between revisions
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==Troubleshooting HLS-Tn5 protocol== | ==Troubleshooting HLS-Tn5 protocol== | ||
* | * AR150527_Tn5_tubeTest | ||
===Motivation=== | ===Motivation=== | ||
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* The results were negative: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Andrew:Notebook/C1_OpenApp_150429 Troubleshooting the Tn5 titration experiment]] | * The results were negative: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Andrew:Notebook/C1_OpenApp_150429 Troubleshooting the Tn5 titration experiment]] | ||
* This indicates that either the protease or Tn5 died, so I am going to make fresh aliquots and repeat. | * This indicates that either the protease or Tn5 died, so I am going to make fresh aliquots and repeat. | ||
* I will also include gDNA from | * I will also include gDNA from ~350 [http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Samples/cells150514 GM12878] cells extracted with HLS + G-HCl and purified with Ampure XP. | ||
* I will use both standard barcoded primers as well as the Nextera transposon sequences. | * I will use both standard barcoded primers as well as the Nextera transposon sequences. | ||
* If the transposon sequences work, I can do the Tn5 titration on chip with a back-loaded Tn5 curve and common PCR mix, followed by barcoded PCR off-chip. | * If the transposon sequences work, I can do the Tn5 titration on chip with a back-loaded Tn5 curve and common PCR mix, followed by barcoded 2nd PCR off-chip. | ||
===Samples=== | |||
* 6 samples total, 3 samples per protocol | |||
* The products from reactions 1-3 will serve as template for reactions 4-6, similar to the C1 protocol | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Condition''' | |||
| align="center" style="background:#f0f0f0;"|'''1''' | |||
| align="center" style="background:#f0f0f0;"|'''2''' | |||
| align="center" style="background:#f0f0f0;"|'''3''' | |||
| align="center" style="background:#f0f0f0;"|'''4''' | |||
| align="center" style="background:#f0f0f0;"|'''5''' | |||
| align="center" style="background:#f0f0f0;"|'''6''' | |||
|- | |||
| Protocol||Transposon PCR||Transposon PCR||Transposon PCR||Barcode 2nd PCR||Barcode 2nd PCR||Barcode 2nd PCR | |||
|- | |||
| Sample||350 GM12878||Purified DNA||NTC||350 GM12878||Purified DNA||NTC | |||
|- | |||
| | |||
|} | |||
===Protocol=== | |||
* I got a fresh aliquot of 20 mg/mL Qiagen Protease from Brandon. | |||
* Adjustment factors: | |||
** HLS ProtQ mix --> 1.5X | |||
** Tn5 --> 1.75X | |||
** ProtQ --> 1.2X | |||
** PCR --> 2.2X | |||
Transposon primed 1st PCR | |||
# Start with 1 uL: Cells, DNA, or NTC | |||
# Make HLS ProtQ mix for 5 reactions, 2 uL each, 1.5X: 10 uL of 7.5 mg/mL ProtQ, 45 mM Tris-HCl | |||
## 3.75 uL 20 mg/mL ProtQ | |||
## 1.125 uL 400 mM Tris-HCl | |||
## 5.125 uL H2O | |||
## Longer lysis and heat-kill: 30 min at 50 C and 30 min at 70 C. | |||
# Make Tn5 mix for 4 reactions, 4 uL each, 1.75X: 16 uL of 0.15X Tn5 in Tn5 diluent | |||
## 2.4 uL 1X Tn5 | |||
## 13.6 uL Tn5 diluent | |||
# Make ProtQ mix for 4 reactions, 30 uL each, 1.2X: 120 uL of 0.24 mg/mL ProtQ, 30 mM Tris-HCl | |||
## 1.44 uL 20 mg/mL ProtQ | |||
## 9 uL 400 mM Tris-HCl | |||
## 109.5 uL H2O | |||
# Make PCR mix for 4 reactions, 30 uL each, 2.2X: 120 uL of 2.2X NPM, 440 nM each primer | |||
## 79 uL 3.33X NPM | |||
## 5.28 uL 10 uM Nextera P7 transposon | |||
## 5.28 uL 10 uM Nextera P5 transposon | |||
## 30.44 uL H2O | |||
* Used 68 degree annealing temp | |||
Barcoded 2nd PCR | |||
# 3 uL 1st PCR product | |||
# 1 uL 10 uM N7 primer | |||
# 1 uL 10 uM S5 primer | |||
# 5 uL 2X KAPA SYBR Fast | |||
===Gels=== | |||
[[File:AR150527_gels_Slide1.jpg|600px]] | |||
[[File:AR150527_gels_Slide2.jpg|600px]] |
Latest revision as of 19:27, 31 May 2015
Troubleshooting HLS-Tn5 protocol[edit]
- AR150527_Tn5_tubeTest
Motivation[edit]
- My last experiment tested HLS+ProtQ, Tn5-059, ProtQ digestion, and PCR with standard Nextera XT v2 barcoded primers.
- The results were negative: [Troubleshooting the Tn5 titration experiment]
- This indicates that either the protease or Tn5 died, so I am going to make fresh aliquots and repeat.
- I will also include gDNA from ~350 GM12878 cells extracted with HLS + G-HCl and purified with Ampure XP.
- I will use both standard barcoded primers as well as the Nextera transposon sequences.
- If the transposon sequences work, I can do the Tn5 titration on chip with a back-loaded Tn5 curve and common PCR mix, followed by barcoded 2nd PCR off-chip.
Samples[edit]
- 6 samples total, 3 samples per protocol
- The products from reactions 1-3 will serve as template for reactions 4-6, similar to the C1 protocol
Condition | 1 | 2 | 3 | 4 | 5 | 6 |
Protocol | Transposon PCR | Transposon PCR | Transposon PCR | Barcode 2nd PCR | Barcode 2nd PCR | Barcode 2nd PCR |
Sample | 350 GM12878 | Purified DNA | NTC | 350 GM12878 | Purified DNA | NTC |
Protocol[edit]
- I got a fresh aliquot of 20 mg/mL Qiagen Protease from Brandon.
- Adjustment factors:
- HLS ProtQ mix --> 1.5X
- Tn5 --> 1.75X
- ProtQ --> 1.2X
- PCR --> 2.2X
Transposon primed 1st PCR
- Start with 1 uL: Cells, DNA, or NTC
- Make HLS ProtQ mix for 5 reactions, 2 uL each, 1.5X: 10 uL of 7.5 mg/mL ProtQ, 45 mM Tris-HCl
- 3.75 uL 20 mg/mL ProtQ
- 1.125 uL 400 mM Tris-HCl
- 5.125 uL H2O
- Longer lysis and heat-kill: 30 min at 50 C and 30 min at 70 C.
- Make Tn5 mix for 4 reactions, 4 uL each, 1.75X: 16 uL of 0.15X Tn5 in Tn5 diluent
- 2.4 uL 1X Tn5
- 13.6 uL Tn5 diluent
- Make ProtQ mix for 4 reactions, 30 uL each, 1.2X: 120 uL of 0.24 mg/mL ProtQ, 30 mM Tris-HCl
- 1.44 uL 20 mg/mL ProtQ
- 9 uL 400 mM Tris-HCl
- 109.5 uL H2O
- Make PCR mix for 4 reactions, 30 uL each, 2.2X: 120 uL of 2.2X NPM, 440 nM each primer
- 79 uL 3.33X NPM
- 5.28 uL 10 uM Nextera P7 transposon
- 5.28 uL 10 uM Nextera P5 transposon
- 30.44 uL H2O
- Used 68 degree annealing temp
Barcoded 2nd PCR
- 3 uL 1st PCR product
- 1 uL 10 uM N7 primer
- 1 uL 10 uM S5 primer
- 5 uL 2X KAPA SYBR Fast