h***9
发帖数: 662 | 1 一个准备用来做knock-in老鼠的targeting vector（14kb),用invitrogen的kit做了
maxi-prep,然后用NotI linearize,我确定NotI是单一酶切位点,可是为什么出来这么多
大大小小的片断（4kb,2.5kb,1.5kb,100bp)？ |
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Z******5
发帖数: 435 | 2 多谢楼上两位。
病毒最后包装的部分应该是HIV-5'LTR到HIV-3'LTR之间的部分吧。pll3.7载体上这部分
大小是5kb，我改造后大小是6kb。我要分别构建两个转录因子的载体，一个是500bp，
另一个1.4kb。我想应该没什么问题。 |
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l******o
发帖数: 103 | 3 I have the following problem. I want to introduce a point mutation to my
plasmid. The plasmid is super big. So I first cut out a fragment(~4kb) with
XbaI and then ligate it to another vector. After introducing mutation with
mutagenesis kit, the sequencing result is as expected.
Next step is to put the XbaI-XbaI fragment containing the mutation back
into the original clone. After ligation and transformation with DH5alpha I
get a number of colonies (~20) but no colonies in the negative control. B... 阅读全帖 |
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e****s
发帖数: 1125 | 4 I would check plasmid and expression more carefully before draw a conclusion
of genomic recombination.
1. Have you check the whole 4kb cDNA after site-direct mutagenesis? It will
take you 5 sequencing to check the whole cDNA.
2. How do you check the expression? Have you check both supernatant and
total lysis? by western or just SDS-PAGE. The point is single mutant could
dramatically decrease the expression.
3. Purify plasmid from the expression E.coli, do cutting test and send for
sequencing.
w... 阅读全帖 |
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u*********1
发帖数: 2518 | 5 Illumina Hiseq2000 I guess is mainstream device
But seems Hiseq2500 is coming out.
Also if you're interested in very long sequences (say, ~4kb) for assembly
study, then PacBio though with a relatively higher error rate.
I'm not an expert in this and also would like to hear more |
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d**p
发帖数: 149 | 6 4kb is not a 大片段. Just regular cloning |
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b*********e
发帖数: 870 | 7 infusion 效率非常高的，根本不用操心什么浓度。我们把整个infusion反应体系压缩
到2ul，就是只用0.4ul infusion mix，剩下的1.6ul就是vector和inserts的回收产物
分配一下。
再说了，你这个4kb也不长啊，你跑胶回收的时候，把洗脱buffer加热一下，洗脱效率
高很多的。 |
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w*****t
发帖数: 179 | 8 我用的是clonetech的INFUSION，载体4KB，连了4个片段进去，是在一个反应，最后大
小11KB，挑了4个有两个阳性。不是很复杂，但是设计引物的时候要费点事，注意5’，
3‘设计的时候不要搞错。 |
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y******8
发帖数: 1764 | 9 这个更像pacbio的技术。但是要好很多。测conductivity可以避免现在pacbio的致命缺
陷。
从原理上看精准度应该和现有的nanopore差不多，但也可能好上一个数量级。毕竟合成
的速度比单纯的过一个孔要慢很多。如果现在nanopore是raw error rate 4%, 这个应
该在0.1 - 5%.
从读长上应该和pacbio差不多，都是取决于polymerase的processability. 500bp-4kb
左右吧，但是可以宣称做到10-20kb。 |
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C***H
发帖数: 508 | 10 是的，很像pacbio，误码率低了不止一个数量级，原理上读长应该差不多，不知道为什
么他们只用50mer。
另外一个奇怪的地方是他们加了9V的电压在IgG的两臂上。溶液里加这么高的电压完全
不make sense... 就算没问题，基本上一个电子transfer的能量就是9eV或者4.5eV，可
比pacbio的光子能量大多了，不知道会不会损伤蛋白。
4kb |
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C******2
发帖数: 97 | 11 求助各位，最近我在操作一个逆转录病毒载体，在两个ltr 之间插入了大概 4kb 的片
断 ，两个ltr呈反向排列，但是之间只有20bp的间隔，具体情况见下图，但是最终不能
正常产生病毒，请教各位有什么好的建议么，或者说这样的载体本身就不能正常工作？
谢谢大家的建议。 |
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B******o
发帖数: 496 | 12 你的载体是啥？具体说说两个LTR之间的序列，通常会有些包装蛋白的element。
另外，4kb算是比较大得了，病毒的产量会随insert 长度有指数的下降 |
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H*******i
发帖数: 196 | 13 各种方法都有优缺点，合理使用才是关键
如果要构建大量同系列克隆，酶切连接自然有优势（至少不用成天买primer），而且在
最后成环一步，酶切连接也是最robust的方法之一（有时二级结构对Gibson/Slic有点
影响，尤其是一律50°C的反应，基于PCR和recombination的问题就更多了。） Scar
只要用overlapping PCR解决就是了。
当然完全构建一个全新的，一次性的需要大量不同片段的东西，自然Gibson assembly
类方法有优势（做特别大的片段也有优势）
Gibson得话 T5 exonuclease https://www.neb.com/products/m0363-t5-exonuclease
Taq ligase https://www.neb.com/products/m0208-taq-dna-ligase
加上polymerase
就两三刀吧。
Slic一样片段，只用T4 DNApolymerase，两片段效率大概低两个数量级。
另外LZ你需不需要额外纯化是根据你片段性质来得，你7kb的是啥？ insert得话必须
纯化，无论什么... 阅读全帖 |
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Z******5
发帖数: 435 | 14 invitrogen的superscript3 kit中的反应时间是40min，说明书中说4kb以内的片段都没
问题。 我之前有做过13kb的，延伸2个小时，也能搞定。 |
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Z******5
发帖数: 435 | 15 用invitrogen的这个kit
5' RACE System for Rapid Amplification of cDNA Ends, version 2.0
说明书上说貌似50min反转录4kb没问题。 我之前做大约10kb的，反转录2个小时，结果
很好。 不过我那个基因表达丰度非常高。如果表达丰度很低的，不知道效果会如何。
另外，建议在预测的TSS后面1-2kb的地方同时设计一个引物做5'RACE， 看看和你从
11kb的位置拉出来的是不是同一个起始位点。 |
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Z******5
发帖数: 435 | 16 我记得很久以前看过一个protocol，貌似说反转录60分钟，4kb的没有问题。 我自己做
都是搞120分钟，这个应该没啥坏处。
以前搞过一个6kb多的epigenetic factor，真心不好搞，最后分了4段做overlap PCR才
搞出来了。如果你的目的基因丰度又低，片段又长，真的会很难搞。 最好买个cDNA回
来搞吧。 如果实在不想花那么多钱，做overlap也可以，但是不要搞酶切位点的链接，
用同源重组的链接，这样设计引物自由度会大很多。 |
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w*******y
发帖数: 60932 | 17 2.5" Solid State Drives / SATA II / MLC / Performance Series / 60GB -
OCZSSD2-2VTXE60G
Max Read: up to 285MB/s
Max Write: up to 275MB/s
Sustained Write: up to 250MB/s
Random Write 4KB (Aligned): 50,000 IOPS
Native TRIM support
Max IOPS Firmware
Seek Time: 0.1ms
Slim 2.5" Design
99.8 x 69.63 x 9.3mm
Lightweight: 77g
Operating Temp: 0C ~ 70C
Storage Temp: -45C ~ +85C
Low Power Consumption: 2W in operation, 0.5W in standby
Shock Resistant up to 15 |
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w*******y
发帖数: 60932 | 20 New Sata 3 120 gb SSD very fast: Link:
http://www.newegg.com/Product/Product.aspx?Item=N82E16820227729
Features:
Max Read up to 500MB/s Max Write up to 450MB/s 4KB Random Write: 20,000
IOPS Up to 55 bits correctable per 512-byte sector (BCH) varies depending on
exact configuration Self-Monitoring, Analysis and ReportingTechnology (S.M.
A.R.T.) Fully compliant with Serial ATA InternationalOrganiz ation: Serial
ATA Revision 3.0.Fully compliant with ATA/ATAPI-8StandardNative Command
Queuing (N... 阅读全帖 |
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w*******y
发帖数: 60932 | 21 OCZ VTX3-25SAT3-120G Vertex 3 Solid State Drive - 120GB, 2.5", SATA III
$199.99 after $30 Mail in rebate
MIR Expires 7/4
This is the cheapest I've seen the Vertex 3 at. This is still the fastest
SATA III SSD on the market.
Specs:
120GB Max Performance*
Max Read: up to 550MB/s
Max Write: up to 500MB/s
Random Write 4KB: 60,000 IOPS
Maximum 4K Random Write: 85,000 IOPS
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w*******y
发帖数: 60932 | 22 OCZ Agility 3 240GB 2.5IN SATA3 Sandforce SF-2281 SSD Solid State Disk Flash
Drive
Max Read: up to 525MB/s
Max Write: up to 500MB/s
Random Write 4KB (Aligned): 45,000 IOPS
240GB
MLC NAND Flash
Interface: SATA 6Gbps / Backwards Compatible 3Gbps
Native TRIM support
Seek Time: .1ms
Slim 2.5" Design
99.8 x 69.63 x 9.3mm
Lightweight: 77g
Operating Temp: 0癈 ~ 55癈
Storage Temp: -45癈 ~ 85癈
Low Power Consumption: 2.7W Active, 1.5W Idle
Shock Resistant up to 1500G
RAID Support
Compatible with Windows 7, V... 阅读全帖 |
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w*******y
发帖数: 60932 | 23 Link:
http://us.ncix.com/products/?sku=60773&vpn=OCZSSDPX-1RVD0230&ma Technology&promoid=1316
230GB
PCI-Express interface (x4)
For use as primary boot drive or data storage
Internal RAID 0
181.07 (L) x 21.59 (W) x 125.08mm (H)
Shock Resistance: 1500g
Seek Time: 0.1 ms
Operating Temp: 0癈 ~ 70癈
Storage Temp: -45癈 ~ 85癈
Power Consumption: 3W Idle, 8W active
MTBF: 2,000,000 hours
3-Year Warranty
Compatible with Windows XP 32/64, Vista 32/64, Window... 阅读全帖 |
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w*******y
发帖数: 60932 | 24 Not really a deal, but this may help you get burned on a supposed "good deal
" and the device
I see a lot of "Class XX" bashing on these forums when deals are posted for
microSD cards. Class ratings only state a MINIMUM sequential read speed and
nothing else (which may be fine for cameras, but it isn't for phones).
Routinely a lot of class 2s and class 4s will benchmark at class 10 speed or
higher.
A lot of the users over at the XDA Devs forums (especially the nook color
selection, due to the s... 阅读全帖 |
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w*******y
发帖数: 60932 | 25 Corsair Force Series 3 CSSD-F120GB3A-BK 2.5" 120GB SATA III Internal Solid
State Drive (SSD)
$159.99 - $30 rebate - $10 coupon = $119.99 shipped
register a new account use coupon NEWCUSTOMER10 for $10 off.
120GB SSD:
http://www.newegg.com/Product/Product.aspx?Item=N82E16820233206
Interface
SATA III
Max Sequential Read
Up to 550 MB/s
Max Sequential Write
Up to 510 MB/s
4KB Random Write
Up to 85,000 IOPS
MTBF
2,000,000 hours
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w*******y
发帖数: 60932 | 27 Mushkin MKNSSDCR120GB Link:
http://www.neweggbusiness.com/Product/Product.aspx?Item=N82E168
$129.99 - ($20 after $100 Coupon) = $109.99
quote
Performance
Max Sequential Read - Up to 550MB/s
Max Sequential Write - Up to 515MB/s
4KB Random Write - Up to 90,000 IOPS
Seek Time - 0.1 ms
MTBF - 2,000,000 hours
Includes an adapter for 3.5" bay.
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