FAQs for PCR & Cloning
Q1) How to ship purified gDNA for PCR?
Ans: We recommend our customers to ship gDNA at 4°C or blue ice to retain the best integrity of the DNA and minimize the risk of sample degradation. Although some orders were received in room temperature without much problem, but the customers whom bear such risk has to accept the proviso charges when the PCR fails due to sample degradation.
Q2) I have a plasmid and I want to keep it as bacterial glycerol stock/slant agar culture. What service should I order?
Ans: You may order plasmid preparation services with additional request of having glycerol stock/ slant agar. Glycerol stock has to be shipped with dry ice and slant agar has to be shipped with blue ice. You may want to consider the additional shipping cost with such conditions.
Q3) If my PCR product is a mixture, which DNA cloning service is suitable?
Ans: You may order MBS-3006, PCR Product cloning Service PLUS (up to 1.5 kb). The PCR product will be cloned into a holding vector before sequencing. The colony PCR products will then submitted for both forward and reverse sequencing. Glycerol stock of any clone can be ordered using our miniprep services (Product no.: MBS-4001 and MBS-4005).
Q4) My insert gene size is more than 1.5 kb, can you still clone them?
Ans: Yes. We can clone the PCR products or DNA fragment larger than 1.5kb. There will be surcharges to sequence the large fragment for sequencing verification. For insert size of 2 kb, the services will thus include MBS-3002, PCR Product Cloning (up to 1.5 kb) and MBS-8001, Primer Walking (charge per base).
Q5) I have clones with various inserts at size > 5 kb, are you able to sequence them?
Ans: If the insert is pure isolate, primer-walking using Sanger sequencing is still good to go. If the insert is expected to be a mixture of similar size, we can offer sequencing using NGS methods.
Q6) I wish to clone my PCR products into an expression vector. Can you do that?
Ans: We recommend cloning the PCR product into a holding vector to verify the insert sequence first. Then you may submit the cloned DNA and your choice of expression vector for our subcloning service. However, we also accept un-purified PCR products at an additional charge.
Q7) Do you perform quality check using restriction enzyme (RE) for your DNA cloning services?
Ans: We verify insert sequence using sequencing by default. If you need DNA digestion using RE, we can offer this as one of the sample preparation service (Product No. MBS-6002).
Q8) My PCR product contains two close bands, should I buy one or two PCR product cloning services?
Ans: When two DNA bands have less than 50 bp of difference in size, we cannot guarantee to separate and cut each individual band using gel extraction successfully. You may order MBS-3006, PCR Product cloning Service PLUS (up to 1.5 kb) for such mixture of PCR products. The PCR product will be cloned into a holding vector before sequencing. 5 of the colony PCR products will then submitted for both forward and reverse sequencing.
Q9) Can I sequence more colonies from your standard positive colony screening?
Ans: Our standard DNA cloning service will sequence only 1 positive clone. You may order another 24 clones to be screened, follow by plasmid extraction for sequencing according to your instruction. We recommend such decision to be made within 2 weeks. The colonies on agar plate will degrade after 2 weeks at 4oC of storage and not recoverable.
Q10) I ordered bacterial glycerol stock. However, I receive it in the liquid condition and they are not frozen. Can I still use it?
Ans: You should not use the glycerol stock. The cells usually die when the stock is left at room temperature for a while.
Q11) The bacterial glycerol stock of my subcloning culture does not work. I cannot obtain bacteria when I streak the bacteria on agar plate.
Ans: The bacteria might be low in number or weak in growing. We usually keep one replicate of glycerol stock for our subcloning products (excludes PCR product cloning) in freezer for one year after the project completes. We can make replacement for you if we still have them. Alternatively, you can inoculate the bacteria in broth and grow it overnight. Then, you can make new glycerol stock.
Q12). My freezer breaks down and the bacterial glycerol stock turns bad.
Ans: We recommend to keep both purified plasmid and glycerol stock in freezer. You still can transform the plasmid if the glycerol stock turns bad. Furthermore, always make new glycerol stock after a few freeze-thaw cycles (at least once a year), which depends on your frequency of usage.
Q13) I don’t have -80oC freezer. Can I store your provided bacterial glycerol stock at -20oC freezer?
Ans: We mix overnight bacterial culture with equal volume of 50% glycerol. The bacterial glycerol stock from -20oC freezer can be used but we do not guarantee its shelf-life.
Q14) I want to order gene synthesis for subcloning. What other concerns should I pay attention?
Ans: There are two: (1) restriction enzymes for subcloning to be included in gene synthesis; (2) the necessity of codon optimization.
Q15) I found some nucleotides change in the insert sequence after subcloning service is completed.
Ans: We encourage customer to verify the original insert sequences from the holding vector before subcloning starts. If the change is verified to happen in the original insert sequence, you may order site-directed mutagenesis to correct the error.
Q16) I want to perform in vitro transcription. What factors to be considered?
Ans: We provide pJET1.2/Blunt from Thermo Fisher Scientific as the default holding vector for our PCR Product Cloning services. It includes T7 RNA polymerase promoter for in vitro transcription of the cloned insert. When you order the service, you may indicate the sense or antisense of transcripts. The sense strand transcripts are used in expression, structural or functional studies. It can also be used to construct a standard curve for RNA quantification using an artificial sense strand of RNA. In contrast, the complementary antisense transcripts are required when they are used as a probe to messenger RNA (mRNA) in Northern blots, in situ hybridizations, and nuclease protection assays.
If you wish to incorporate T7 promoter in your gene synthesis product before subcloning, we can help you in such design before you place the order. Please enquire.
Q17) Why plasmid extracted from BL21(DE3) E.coli cells shows inconsistent DNA patterns when cut by restriction enzymes?
Ans: The BL21(DE3) has endA and recA genes and hence the endonuclease 1 activity is present. The produced plasmid could be low yield or bad in quality. Thus, BL21(DE3) E.coli cells is recommended only for protein expression works.
Q18) I have ordered PCR amplification and followed by PCR product cloning services. Why the inserted gene sequence is not 100% identical to the reference gene from NCBI database?
Ans: The differences found in gene sequence is due to the presence of single nucleotide polymorphisms (SNPs). These are common type of genetic variation among organisms. If you want 100% identical sequence as reference gene from any database, you may consider to order synthetic gene directly.
Q19) I want to amplify genes from fungal samples. What preliminary check should I perform?
Ans: We recommend you to perform DNA barcoding to verify the identity of the fungal sample. This can boost your confidence in carrying out the subsequent lab works.