Brandon:LabNotes/Project1/2012-6-6: Difference between revisions
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*For Mg++ fragmentation protocol see: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-6-15 Magnesium fragmentation protocol]] | *For Mg++ fragmentation protocol see: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-6-15 Magnesium fragmentation protocol]] | ||
*For RNA fragmentation information see [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-5-30#RNA_fragmentation RNA fragmentation info] | *For RNA fragmentation information see [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-5-30#RNA_fragmentation RNA fragmentation info] | ||
*T7-top2 transposon was used. | |||
Line 48: | Line 48: | ||
1. Retrieve IVT RNA. | 1. Retrieve IVT RNA. | ||
*samples: | *samples: | ||
1. with Blocking Primer, protease treated | |||
2. with blocking primer, EtOH cleaned | |||
NTC | 3. NTC with blocking primer, protease | ||
4. without blocking primer, protease treated | |||
5. without blocking primer, EtOH cleaned | |||
6. NTC without blocking primer, protease treated | |||
used 5 uL RNA for each sample | |||
2. Perform EtOH cleanup of RNA. | 2. Perform EtOH cleanup of RNA. (can do columns) | ||
3. Perform RNase III fragmentation (NEB): | 3. Perform RNase III fragmentation (NEB): | ||
*!!!!!8/5/2012 update, used T7-frag-block which is designed for T7-top3!!!!!! (but still worked) | |||
*!!!!!8/9/2012 update, T7-frag-block accidentally used at a concentration of 100 um!!!! | |||
Starting Material: Purified mRNA (50–250 nanograms) | Starting Material: Purified mRNA (50–250 nanograms) | ||
1. Add 5' end blocking DNA primer and do annealing to form DNA-RNA hybrid. | 1. Add 5' end blocking DNA primer and do annealing to form DNA-RNA hybrid. | ||
*a. add 2 uL of blocking oligo (10 uM) to aliquot RNA sample that will be used in step 2. | *a. add 2 uL of blocking oligo (10 uM) to aliquot RNA sample that will be used in step 2. | ||
!!!!!8/5/2012 update, used T7-frag-block which is designed for T7-top3!!!!!! (but still worked) | |||
!!!!!8/9/2012 update, T7-frag-block accidentally used at a concentration of 100 um!!!! | |||
*b. Incubate at 95C for 2 minutes. | *b. Incubate at 95C for 2 minutes. | ||
*c. cool to RT at 0.1 C/s | *c. cool to RT at 0.1 C/s | ||
Line 71: | Line 79: | ||
X uL Purified mRNA + blocking primer (50-250 nanograms) | X uL Purified mRNA + blocking primer (50-250 nanograms) | ||
.5 uL RNase III (1 unit/μl) | |||
1 uL RNase III Reaction Buffer (10X) | |||
5.5 uL Nuclease-Free Water | |||
add in DNA primer to protect 5' end since don't want degradation?? | add in DNA primer to protect 5' end since don't want degradation?? | ||
_______ | _______ | ||
10 uL total volume | |||
Line 114: | Line 122: | ||
*Run TBU gel to see fragmentation profile | |||
Line 144: | Line 153: | ||
3. Add the following to the reaction. | 3. Add the following to the reaction. | ||
2 uL 5X first strand buffer | |||
3 uL dNTP mix | |||
3 uL 100 uM DTT | |||
.75 uL SMART MMLV RT and mix (ADD LAST!!!!!) | |||
____ | ____ | ||
32.5 uL total | |||
4. Incuvate at 42C for 60 minutes | 4. Incuvate at 42C for 60 minutes | ||
Line 163: | Line 172: | ||
25 uL KAPA SYBR | 25 uL KAPA SYBR | ||
4 uL primers, 1 uL F, 1 uL R (T7-top2-PCR or T7-top3-PCR) and (PCR_R.N2Ind[XX] (23,24)) | 4 uL primers, 1 uL F, 1 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX] (23,24)) | ||
1 uL H2O | 1 uL H2O | ||
20 uL DNA template (use whole RT reaction) | 20 uL DNA template (use whole RT reaction) | ||
Line 177: | Line 186: | ||
*run qiaquick to clean sample before performing qPCR. | *run qiaquick to clean sample before performing qPCR. | ||
**6 enzymes from 6 different reactions in there already. | **6 enzymes from 6 different reactions in there already. | ||
===Results=== | ===Results=== | ||
Line 232: | Line 238: | ||
|} | |} | ||
[[File:ZhangLab 2 2012-06-13 19hr 06min-labeled.jpg|600px]] | [[File:ZhangLab 2 2012-06-13 19hr 06min-labeled.jpg|600px]] | ||
*Continuing with cloning and transformation etc. to check if has good genomic DNA inserts | |||
*For ligation, Used 3 uL DNA for sample 1. Used 1 UL of DNA for the other samples since they have 5 ng/ul already | |||
*send in sample 1,7,18,20,21,22,23 for sequencing, could be insert, just very low efficiency | |||
*PCRed 11 white colonies for each sample | |||
*cloning insert information: | |||
see [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-5-7 information for sequences of top2] for sequences | |||
85 bp = size of end with polyA, adaptor 2 (3' end) | |||
46+15 bp = size of 5' end of transposon (+15 since ME sequence is still after primer) (5' end) | |||
_______ | |||
146 | |||
M13 Priming sites | |||
89 bp until insertion from M13 reverse (top strand, is fwd in sequencing) | |||
111 bp until insertion from M13 Forward (bottom strand, is reverse in sequencing) | |||
____ | |||
200 bp | |||
Thus if 200+146 = 346 bp or larger fragment after PCR amplification, then it has an insert. For size selected | |||
insert, needs to be 346+250 = 596 bp to contain a correctly size selected insert. | |||
M13 Reverse sequence (beginning) | |||
[CAGGAAACAGCTATGAC]CATGATTACGCCAAGCTTGGTACCGAGCTCGTC[GGATCCACTAGTAAC]GGCCGCCAGTGTGCTGGAATTCGGCTT | |||
M13 reverse primer usually starts here for sequencing | |||
M13 Forward sequence (end) (plasmid sequence) | |||
GCCGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGCCC | |||
TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTT | |||
ACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC | |||
GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCG | |||
CGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCG | |||
CTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT | |||
TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTA | |||
GTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGAC | |||
TCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGC | |||
CGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATTC | |||
ANGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCG | |||
GATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGAT | |||
*1. with Blocking Primer, protease treated | |||
[[File:ZhangLab 2 2012-06-15 16hr 13min-labeled.jpg|600px]] | |||
*2. with blocking primer, EtOH cleaned | |||
[[File:ZhangLab 2 2012-06-15 16hr 15min-labeled.jpg|600px]] | |||
*4. without blocking primer, protease treated | |||
[[File:ZhangLab 2 2012-06-15 16hr 36min-labeled.jpg|600px]] | |||
*5. without blocking primer, EtOH cleaned | |||
[[File:ZhangLab 2 2012-06-15 16hr 38min-labeled.jpg|600px]] | |||
*2nd set | |||
*for ligation, used 4x the amount of DNA. used 4 UL per tube, opposed to the 1 uL per tube in first transformation. will see if results differ. | |||
*send in samples 2,4,6,9,14,17 for sequencing. few looks like could have a good insert. | |||
*PCR'ed 8 colonies for each sample | |||
*1. with Blocking Primer, protease treated | |||
[[File:ZhangLab 2 2012-06-17 17hr 58min-labeled.jpg|600px]] | |||
*2. with blocking primer, EtOH cleaned | |||
[[File:ZhangLab 2 2012-06-17 17hr 59min-lableled.jpg|600px]] | |||
*4. without blocking primer, protease treated | |||
[[File:ZhangLab 2 2012-06-17 18hr 11min-labeled.jpg|600px]] | |||
*5. without blocking primer, EtOH cleaned | |||
[[File:ZhangLab 2 2012-06-17 18hr 12min-labeled.jpg|600px]] | |||
*CLoning stats: | |||
**1 polyT protease treated confirmed | |||
**1 protease treated genomic insert confirmed | |||
**2 polyT EtOH cleaned confirmed | |||
**3 EtOH cleaned genomic insert confirmed | |||
**table of possibles | |||
**EtOH cleaned with BP, 3 validated genomic inserts (round 1) | |||
**protease treated without BP, 1 validated insert (round 2) | |||
**EtOH cleaned with BP, 2 stopped after polyT (probably are correct) (round2) | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''first round''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''BP''' | |||
| align="center" style="background:#f0f0f0;"|'''No BP''' | |||
|- | |||
| ||protease||3||1 | |||
|- | |||
| ||EtOH||3||0 | |||
|- | |||
| |||||| | |||
|- | |||
| |||||| | |||
|- | |||
| second round||||BP||No BP | |||
|- | |||
| ||protease||2||0 | |||
|- | |||
| ||EtOH||3||1 | |||
|- | |||
| |||||| | |||
|- | |||
| |||||| | |||
|- | |||
| ||||11||2 | |||
|- | |||
| | |||
|} | |||
*Blocking primer is definetly helping, since that is the only difference between the two. EtOH cleanup more efficient than protease treatment in results. | |||
*4 have verified inserts, matches 100% to human genome | |||
**except 1 which has some SNPs since insert is flipped and polyA sequence is sequenced first in the sequencing run, thus disrupting reads after. | |||
*5 are disrupted since polyT is in reverse orientation so harder to sequence, but prolly have viable inserts based on size from PCR gel. | |||
*Not sure why the cloning isn't working as well. Could be bad previous amplifications or TA tailing. Can take PCR amplified DNA, add taq polymerase 2X, at 72C for 30 minutes to add A tails for sure, then beads purification, and cloning/transformation again. | |||
===more results=== | |||
*more results for Rnase III fragmentation etc. [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:LabNotes/Project1/2012-6-21 RNase III, purification, taq2X extension, transformation/cloning etc] | |||
===Notes and primers used etc=== | ===Notes and primers used etc=== | ||
Blocking primer for protecting 5' end from RNase III degradation | |||
T7-frag-block | |||
5'- CTGTCTCTTATACACATCTTAATGTCTCCC/3InvdT/ | |||
3InvdT so don't get any nonspecific extension by MMLV RT. | |||
Stuff used for RNA-seq for BENG160 class | Stuff used for RNA-seq for BENG160 class | ||
Line 267: | Line 407: | ||
5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=86.59) | 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=86.59) | ||
ILA adaptor blue Top2-5’end (T7-top2-PCR) Tm=66.79 | ILA adaptor blue Top2-5’end (T7-top2-PCR-iaf) Tm=66.79 | ||
(can be used if top1 is used for transposition) | |||
Line 273: | Line 414: | ||
5’- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3’ (Tm=81.40) | 5’- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3’ (Tm=81.40) | ||
ILA adaptor blue Top3-5’end (T7-top3-PCR) Tm=60.71 | ILA adaptor blue Top3-5’end (T7-top3-PCR-iaf) Tm=60.71 |
Latest revision as of 01:54, 6 December 2012
RNA fragmentation protocols, RNase III, ([see Mg++ protocol here])[edit]
- For Mg++ fragmentation protocol see: [Magnesium fragmentation protocol]
- For RNA fragmentation information see RNA fragmentation info
- T7-top2 transposon was used.
- After IVT with T7, RNA generated and needs to be fragmented.
- protocol for IVT RNA -> RNase III fragmentation -> PolyA polymerase (MMLV buffer) -> MMLV RT -> PCR amplification
- possible issues
- run with and without 5' blocking primer for DNA-RNA hybrid.
- might need to purify after T7 IVT, since [Tris Hcl] is so much higher.
1X T7 buffer: 400 mM Tris Hcl 8 mM MgCl2 2 mM spermidine-Hcl 25 mM NaCl PH 7.9 1X NEBNext RNase III Reaction Buffer: 10 mM Tris-HCl 10 mM Mg(Cl)2 1 mM DTT 60 mM NaCl pH 8.3 @ 25°C 10X Poly(A) Polymerase buffer: 500 mM Tris-HCl 2.5 M NaCl 100 mM MgCl2 pH 7.9 @ 25°C MMLV Invitrogen (though using clontech) 5X First-Strand Buffer 250 mM Tris-HCl (pH 8.3 at room temperature 375 mM KCl 15 mM MgCl2 0.1 M DTT
Rnase III protocol[edit]
1. Retrieve IVT RNA.
- samples:
1. with Blocking Primer, protease treated 2. with blocking primer, EtOH cleaned 3. NTC with blocking primer, protease 4. without blocking primer, protease treated 5. without blocking primer, EtOH cleaned 6. NTC without blocking primer, protease treated used 5 uL RNA for each sample
2. Perform EtOH cleanup of RNA. (can do columns)
3. Perform RNase III fragmentation (NEB):
- !!!!!8/5/2012 update, used T7-frag-block which is designed for T7-top3!!!!!! (but still worked)
- !!!!!8/9/2012 update, T7-frag-block accidentally used at a concentration of 100 um!!!!
Starting Material: Purified mRNA (50–250 nanograms) 1. Add 5' end blocking DNA primer and do annealing to form DNA-RNA hybrid. *a. add 2 uL of blocking oligo (10 uM) to aliquot RNA sample that will be used in step 2. !!!!!8/5/2012 update, used T7-frag-block which is designed for T7-top3!!!!!! (but still worked) !!!!!8/9/2012 update, T7-frag-block accidentally used at a concentration of 100 um!!!! *b. Incubate at 95C for 2 minutes. *c. cool to RT at 0.1 C/s 2. Mix the following components in a sterile PCR tube: X uL Purified mRNA + blocking primer (50-250 nanograms) .5 uL RNase III (1 unit/μl) 1 uL RNase III Reaction Buffer (10X) 5.5 uL Nuclease-Free Water add in DNA primer to protect 5' end since don't want degradation?? _______ 10 uL total volume 3. Incubate in a preheated thermal cycler for 5 minutes at 37°C. 4. Transfer tube to ice.
4. (EtOH clean up OR RNA column purification) OR protease digestion
- EtOH cleanup
a. Add 80 μl of cold Nuclease-Free water. (brings to 100 ul) b. Add 3 volumes 100% EtOH, .1 volumes 3M NaOAc, and 1/300 volumes glycol Blue to the solution obtained. In this case: 10 uL NaOAc (mix after adding) 300 uL 100% EtOH 1 uL Glycol Blue c. Chill solution in -80 for 30 minutes, cool centrifuge to 4C d. Spin at 4C for 20 minutes at max speed. Chill 75% EtOH. e. Blue pellet should be visible, Discard supernatent. f. Wash the pellet with cold 75% EtOH h. Air dry pellet for up to 10 minutes at room temp to remove residual EtOH j. Resuspend in appropriate volume in nuclease free H2O.
- Protease digestion
To each tube, add: 1 uL Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final []) Incubate: 50C 10 minutes, 70C 20 minutes
- Run TBU gel to see fragmentation profile
5. Poly(A) Addition with polyA polymerase (Enzymatics)
- enzymatics PolyA polymerase.
1. assemble reaction: 2 uL 5X SMART MMLV first strand buffer (rather than 10X polyA buffer) 1 uL polyA enzyme 1 uL 10 mM ATP bring to 10 uL with RNA or w/e 2. Incubate at 37C for 10 minutes 3. Heat inactivate at 70C for 20 minutes. (rui and NEB)
6. single strand synthesis MMLV RT (Clontech)
- Followed protocol for SMART MMLV Reverse Transcriptase
20 uL reaction 1. Add 2.5 uL 20 uM primer stock to RNA sample. Bring to final volume of 11.5 uL with Nuclease free H2O (one from BENG160 class, T20VN_PE_R) 2. heat the mixture to 70C fo 3 minutes. Immediately cool on ice. 3. Add the following to the reaction. 2 uL 5X first strand buffer 3 uL dNTP mix 3 uL 100 uM DTT .75 uL SMART MMLV RT and mix (ADD LAST!!!!!) ____ 32.5 uL total 4. Incuvate at 42C for 60 minutes 5. Terminate the reaction by heating at 70C for 10 minutes
7. second strand synthesis (qPCR) (KAPA)
KAPA SYBR FAST qPCR mix X35 cycles 25 uL KAPA SYBR 4 uL primers, 1 uL F, 1 uL R (T7-top2-PCR-iaf or T7-top3-PCR-iaf) and (PCR_R.N2Ind[XX] (23,24)) 1 uL H2O 20 uL DNA template (use whole RT reaction) KAPA SYBR cycles: 98C 3min, (98C for 30s, 60C for 30s, 72C for 1 min) X35, 72C for 5 min, 4C forever
- terminate when curves saturate
8.qiaquick cleanup
- run on gel or w/e
- run qiaquick to clean sample before performing qPCR.
- 6 enzymes from 6 different reactions in there already.
Results[edit]
- TBU gel after RNase III fragmentation and purification with EtOH or enzyme inactivation with protease
- smears still present
File:ZhangLab 2 2012-06-07 18hr 02min-labled.jpg
- result from PCR amplification
- odd how samples 1,2 and 3,4 have almost the same smear even though only sample 1 had high amplification readings during PCR.
File:2012-06-12 qPCR after rnase III with plate.bmp
- TBE gel after RT, PCR amplification
- smears look good, can size select and proceed to cloning/transformation to check for correct inserts.
File:ZhangLab 2 2012-06-12 16hr 35min-labeled.jpg
- TBU gel after RT, PCR amplification
File:ZhangLab 2 2012-06-12 18hr 02min-labeled.jpg
- gel size selection of band, 250 bp to 1000 bp.
File:ZhangLab 2 2012-06-13 15hr 14min-labeled.jpg
- gel after size selection. used 4 uL total of PCR amplified product, resuspended in 10 uL of nuclease free H2O. Have about 50% sample loss from gel size selection. Original samples have a total of 122.28 ng/4 uL, and 102.68 ng/4 uL. Except sample 1, lost alot of sample somehow. probably pipetting or something.
- quanitation from size selection gel.
smpl1 | 0.72 | ng/ul | in 10 uL |
smpl2 | 4.78 | ng/ul | in 10 uL |
smpl4 | 6.08 | ng/ul | in 10 uL |
smpl5 | 4.8 | ng/ul | in 10 uL |
7.2 | ng/total | ||
47.8 | ng/total | ||
60.8 | ng/total | ||
48 | ng/total | ||
File:ZhangLab 2 2012-06-13 19hr 06min-labeled.jpg
- Continuing with cloning and transformation etc. to check if has good genomic DNA inserts
- For ligation, Used 3 uL DNA for sample 1. Used 1 UL of DNA for the other samples since they have 5 ng/ul already
- send in sample 1,7,18,20,21,22,23 for sequencing, could be insert, just very low efficiency
- PCRed 11 white colonies for each sample
- cloning insert information:
see information for sequences of top2 for sequences 85 bp = size of end with polyA, adaptor 2 (3' end) 46+15 bp = size of 5' end of transposon (+15 since ME sequence is still after primer) (5' end) _______ 146 M13 Priming sites 89 bp until insertion from M13 reverse (top strand, is fwd in sequencing) 111 bp until insertion from M13 Forward (bottom strand, is reverse in sequencing) ____ 200 bp Thus if 200+146 = 346 bp or larger fragment after PCR amplification, then it has an insert. For size selected insert, needs to be 346+250 = 596 bp to contain a correctly size selected insert. M13 Reverse sequence (beginning) [CAGGAAACAGCTATGAC]CATGATTACGCCAAGCTTGGTACCGAGCTCGTC[GGATCCACTAGTAAC]GGCCGCCAGTGTGCTGGAATTCGGCTT M13 reverse primer usually starts here for sequencing M13 Forward sequence (end) (plasmid sequence) GCCGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGCCC TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTT ACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC GATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCG CGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCG CTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTT TAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTA GTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGAC TCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGC CGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATTC ANGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCG GATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGCTTGCAGTGGGCTTACATGGCGAT
- 1. with Blocking Primer, protease treated
File:ZhangLab 2 2012-06-15 16hr 13min-labeled.jpg
- 2. with blocking primer, EtOH cleaned
File:ZhangLab 2 2012-06-15 16hr 15min-labeled.jpg
- 4. without blocking primer, protease treated
File:ZhangLab 2 2012-06-15 16hr 36min-labeled.jpg
- 5. without blocking primer, EtOH cleaned
File:ZhangLab 2 2012-06-15 16hr 38min-labeled.jpg
- 2nd set
- for ligation, used 4x the amount of DNA. used 4 UL per tube, opposed to the 1 uL per tube in first transformation. will see if results differ.
- send in samples 2,4,6,9,14,17 for sequencing. few looks like could have a good insert.
- PCR'ed 8 colonies for each sample
- 1. with Blocking Primer, protease treated
File:ZhangLab 2 2012-06-17 17hr 58min-labeled.jpg
- 2. with blocking primer, EtOH cleaned
File:ZhangLab 2 2012-06-17 17hr 59min-lableled.jpg
- 4. without blocking primer, protease treated
File:ZhangLab 2 2012-06-17 18hr 11min-labeled.jpg
- 5. without blocking primer, EtOH cleaned
File:ZhangLab 2 2012-06-17 18hr 12min-labeled.jpg
- CLoning stats:
- 1 polyT protease treated confirmed
- 1 protease treated genomic insert confirmed
- 2 polyT EtOH cleaned confirmed
- 3 EtOH cleaned genomic insert confirmed
- table of possibles
- EtOH cleaned with BP, 3 validated genomic inserts (round 1)
- protease treated without BP, 1 validated insert (round 2)
- EtOH cleaned with BP, 2 stopped after polyT (probably are correct) (round2)
first round | ' | BP | No BP |
protease | 3 | 1 | |
EtOH | 3 | 0 | |
second round | BP | No BP | |
protease | 2 | 0 | |
EtOH | 3 | 1 | |
11 | 2 | ||
- Blocking primer is definetly helping, since that is the only difference between the two. EtOH cleanup more efficient than protease treatment in results.
- 4 have verified inserts, matches 100% to human genome
- except 1 which has some SNPs since insert is flipped and polyA sequence is sequenced first in the sequencing run, thus disrupting reads after.
- 5 are disrupted since polyT is in reverse orientation so harder to sequence, but prolly have viable inserts based on size from PCR gel.
- Not sure why the cloning isn't working as well. Could be bad previous amplifications or TA tailing. Can take PCR amplified DNA, add taq polymerase 2X, at 72C for 30 minutes to add A tails for sure, then beads purification, and cloning/transformation again.
more results[edit]
- more results for Rnase III fragmentation etc. RNase III, purification, taq2X extension, transformation/cloning etc
Notes and primers used etc[edit]
Blocking primer for protecting 5' end from RNase III degradation T7-frag-block 5'- CTGTCTCTTATACACATCTTAATGTCTCCC/3InvdT/ 3InvdT so don't get any nonspecific extension by MMLV RT.
Stuff used for RNA-seq for BENG160 class
5’ end addition primers TSO_N10_BC[XX] [AAGCAGTGGTATCAACGCAGAGdUdU]NNNNNNNNNNTTTAGGrGrGrG adatpor1 5'- AAG CAG TGG TAT CAA CGC AGA G/ideoxyU//ideoxyU/ NNN NNN NNN NTT TAG GrGrGrG -3' P1-STRT 5’- [AATGATACGGCGACCACCGA][GATCT][AAGCAGTGGTATCAACGCAGAGT] -3’ (Tm=82.59) ILA adaptor blue Tm=64.31 adaptor1 Tm=61.02 STRT-SEQ 5'- [GATCT][AAGCAGTGGTATCAACGCAGAGTT] -3' adaptor1
3’ end addition primers T20VN_PE_R 5'-Bio-[GCATTCCTGCTGAACCGCTCTT]CCGATCTTTTTTTTTTTTTTTTTTTTTVN -3’ (Tm=78.92) adaptor2 Tm=65.68 PCR_R.N2Ind[XX] (XX=23,24 for me) 5’- [CAAGCAGAAGACGGCATACGAGAT][TACAAG]CTCG][GCATTCCTGCTGAACCGCTCTT] -3’ (Tm=87.12) ILA adaptor orange bc adaptor2 Tm=65.68
My modifications for T7-tspns for 5' addition primers To use with (T7-top and T7-top2) transposons 5’- [AATGATACGGCGACCACCGA][GATCT][CTCCCTCGCGCCATCAGAGAT] -3’ (Tm=86.59) ILA adaptor blue Top2-5’end (T7-top2-PCR-iaf) Tm=66.79 (can be used if top1 is used for transposition) To use with (T7-top3) transposon 5’- [AATGATACGGCGACCACCGA][GATCT][GGGAGACATTAAGATGTGTATAAGAGACAG] -3’ (Tm=81.40) ILA adaptor blue Top3-5’end (T7-top3-PCR-iaf) Tm=60.71